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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 21 May 2019 at 01:35 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: "CRISPR.CAS" OR "crispr/cas" NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

RevDate: 2019-05-20

Labun K, Montague TG, Krause M, et al (2019)

CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing.

Nucleic acids research pii:5491735 [Epub ahead of print].

The CRISPR-Cas system is a powerful genome editing tool that functions in a diverse array of organisms and cell types. The technology was initially developed to induce targeted mutations in DNA, but CRISPR-Cas has now been adapted to target nucleic acids for a range of purposes. CHOPCHOP is a web tool for identifying CRISPR-Cas single guide RNA (sgRNA) targets. In this major update of CHOPCHOP, we expand our toolbox beyond knockouts. We introduce functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions. We incorporate new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes. Finally, we expand our results page visualization to reveal alternative isoforms and downstream ATG sites, which will aid users in avoiding the expression of truncated proteins. The CHOPCHOP web tool now supports over 200 genomes and we have released a command-line script for running larger jobs and handling unsupported genomes. CHOPCHOP v3 can be found at https://chopchop.cbu.uib.no.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Otoupal PB, Ito M, Arkin AP, et al (2019)

Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides.

mSphere, 4(2): pii:4/2/e00099-19.

Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae Significant advancements in the past few years have bolstered R. toruloides' engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism.IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast's production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Myrbråten IS, Wiull K, Salehian Z, et al (2019)

CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in Lactobacillus plantarum.

mSphere, 4(2): pii:4/2/e00007-19.

Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium.IMPORTANCEL. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Lombardi L, Oliveira-Pacheco J, G Butler (2019)

Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species.

mSphere, 4(2): pii:4/2/e00125-19.

Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalisIMPORTANCECandida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis We have also constructed a separate system for gene editing in C. tropicalis.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Wong E, Liao GP, Chang JC, et al (2019)

GSAP modulates γ-secretase specificity by inducing conformational change in PS1.

Proceedings of the National Academy of Sciences of the United States of America, 116(13):6385-6390.

The mechanism by which γ-secretase activating protein (GSAP) regulates γ-secretase activity has not yet been elucidated. Here, we show that knockout of GSAP in cultured cells directly reduces γ-secretase activity for Aβ production, but not for Notch1 cleavage, suggesting that GSAP may induce a conformational change contributing to the specificity of γ-secretase. Furthermore, using an active-site-directed photoprobe with double cross-linking moieties, we demonstrate that GSAP modifies the orientation and/or distance of the PS1 N-terminal fragment and the PS1 C-terminal fragment, a region containing the active site of γ-secretase. This work offers insight into how GSAP regulates γ-secretase specificity.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Boettcher M, Covarrubias S, Biton A, et al (2019)

Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding.

BMC genomics, 20(1):107 pii:10.1186/s12864-019-5480-0.

BACKGROUND: While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells.

RESULTS: Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA.

CONCLUSION: Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Vicencio J, Martínez-Fernández C, Serrat X, et al (2019)

Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans.

Genetics, 211(4):1143-1154.

CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (≤200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Cornel MC, Howard HC, Lim D, et al (2019)

Moving towards a cure in genetics: what is needed to bring somatic gene therapy to the clinic?.

European journal of human genetics : EJHG, 27(3):484-487.

Clinical trials using somatic gene editing (e.g., CRISPR-Cas9) have started in Europe and the United States and may provide safe and effective treatment and cure, not only for cancers but also for some monogenic conditions. In a workshop at the 2018 European Human Genetics Conference, the challenges of bringing somatic gene editing therapies to the clinic were discussed. The regulatory process needs to be considered early in the clinical development pathway to produce the data necessary to support the approval by the European Medicines Agency. The roles and responsibilities for geneticists may include counselling to explain the treatment possibilities and safety interpretation.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Xu R, Qin R, Li H, et al (2019)

Enhanced genome editing in rice using single transcript unit CRISPR-LbCpf1 systems.

Plant biotechnology journal, 17(3):553-555.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Miao J, Yang Z, Zhang D, et al (2019)

Mutation of RGG2, which encodes a type B heterotrimeric G protein γ subunit, increases grain size and yield production in rice.

Plant biotechnology journal, 17(3):650-664.

Heterotrimeric G proteins, which consist of Gα , Gβ and Gγ subunits, function as molecular switches that regulate a wide range of developmental processes in plants. In this study, we characterised the function of rice RGG2, which encodes a type B Gγ subunit, in regulating grain size and yield production. The expression levels of RGG2 were significantly higher than those of other rice Gγ -encoding genes in all tissues tested, suggesting that RGG2 plays essential roles in rice growth and development. By regulating cell expansion, overexpression of RGG2 in Nipponbare (NIP) led to reduced plant height and decreased grain size. By contrast, two mutants generated by the clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system in the Zhenshan 97 (ZS97) background, zrgg2-1 and zrgg2-2, exhibited enhanced growth, including elongated internodes, increased 1000-grain weight and plant biomass and enhanced grain yield per plant (+11.8% and 16.0%, respectively). These results demonstrate that RGG2 acts as a negative regulator of plant growth and organ size in rice. By measuring the length of the second leaf sheath after gibberellin (GA3) treatment and the GA-induced α-amylase activity of seeds, we found that RGG2 is also involved in GA signalling. In summary, we propose that RGG2 may regulate grain and organ size via the GA pathway and that manipulation of RGG2 may provide a novel strategy for rice grain yield enhancement.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Teng Y, Luo M, Yu T, et al (2019)

CRISPR/Cas9-mediated deletion of miR-146a enhances antiviral response in HIV-1 infected cells.

Genes and immunity, 20(4):327-337.

The human immunodeficiency virus type 1 (HIV-1) causes persistent infection in human and induces miR-146a expression in infected cells. miR-146a represses the innate immune response by inhibiting the expression of TRAF6 and IRAK1 genes, thus negatively controls the NF-κB-related cytokines and interferon stimulated genes. Here we reported that lentiviral CRISPR/Cas9 system was highly efficient in introducing mutations in the precursor miR-146a genomic sequences, resulting in a loss of miR-146a expression and function. miR-146a ablation led to increasing cytokines production in LPS-stimulated A549 cells. Moreover, miR-146a knockout in HIV-1 infected MT2 cells markedly increased the expression of cytokines and HIV-1 restriction factors and reversed T cell exhaustion markers expression, thus influencing HIV-1 replication. Our study indicates that lentiviral CRISPR/Cas9-mediated gene editing is an effective approach to abrogate miR-146a expression, which consequently inhibits HIV-1 replication as well as proviral reactivation by enhancing the expression of cytokines and HIV-1 restriction factors.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Smith PA (2018)

The best Cas scenario.

Nature medicine, 24(5):528-530.

RevDate: 2019-05-20
CmpDate: 2019-05-20

McHugh T, Gluszek AA, JPI Welburn (2018)

Microtubule end tethering of a processive kinesin-8 motor Kif18b is required for spindle positioning.

The Journal of cell biology, 217(7):2403-2416.

Mitotic spindle positioning specifies the plane of cell division during anaphase. Spindle orientation and positioning are therefore critical to ensure symmetric division in mitosis and asymmetric division during development. The control of astral microtubule length plays an essential role in positioning the spindle. In this study, using gene knockout, we show that the kinesin-8 Kif18b controls microtubule length to center the mitotic spindle at metaphase. Using in vitro reconstitution, we reveal that Kif18b is a highly processive plus end-directed motor that uses a C-terminal nonmotor microtubule-binding region to accumulate at growing microtubule plus ends. This region is regulated by phosphorylation to spatially control Kif18b accumulation at plus ends and is essential for Kif18b-dependent spindle positioning and regulation of microtubule length. Finally, we demonstrate that Kif18b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work reveals that Kif18b uses its motile properties to reach microtubule ends, where it regulates astral microtubule length to ensure spindle centering.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Shen S, Sanchez ME, Blomenkamp K, et al (2018)

Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with Genome Editing in Transgenic Mice.

Human gene therapy, 29(8):861-873.

Alpha-1 antitrypsin deficiency (AATD) is a hereditary liver disease caused by mutations in the SERPINA1 serine protease inhibitor gene. Most severe patients are homozygous for PiZ alleles (PiZZ; amino acid E324K), which lead to protein aggregates in hepatocytes and reduced circulating levels of AAT. The liver aggregates typically lead to fibrosis, cirrhosis, and hepatocellular carcinoma, and the reduced circulating AAT levels can lead to emphysema and chronic obstructive pulmonary diseases. In this study, two CRISPR/Cas9 gene editing approaches were used to decrease liver aggregates and increase systemic AAT-M levels in the PiZ transgenic mouse. In the first approach, AAT expression in hepatocytes was reduced more than 98% following the systemic delivery of AAV8-CRISPR targeting exon 2 of hSERPINA1, leading to reduced aggregates in hepatocytes. In the second approach, a second adeno-associated virus, which provided the donor template to correct the Z mutation, was also administered. These treated mice had reduced AAT expression (> 98%) and a low level (5%) of wildtype AAT-M mRNA. Taken together, this study shows that CRISPR gene editing can efficiently reduce liver expression of AAT-Z and restore modest levels of wildtype AAT-M in a mouse model of AATD, raising the possibility of CRISPR gene editing therapeutic for AATD.

RevDate: 2019-05-20
CmpDate: 2019-05-20

Song CQ, Wang D, Jiang T, et al (2018)

In Vivo Genome Editing Partially Restores Alpha1-Antitrypsin in a Murine Model of AAT Deficiency.

Human gene therapy, 29(8):853-860.

CRISPR (clustered regularly interspaced short palindromic repeats) genome editing holds promise in the treatment of genetic diseases that currently lack effective long-term therapies. Patients with alpha-1 antitrypsin (AAT) deficiency develop progressive lung disease due to the loss of AAT's antiprotease function and liver disease due to a toxic gain of function of the common mutant allele. However, it remains unknown whether CRISPR-mediated AAT correction in the liver, where AAT is primarily expressed, can correct either or both defects. Here we show that AAV delivery of CRISPR can effectively correct Z-AAT mutation in the liver of a transgenic mouse model. Specifically, we co-injected two AAVs: one expressing Cas9 and another encoding an AAT guide RNA and homology-directed repair template. In both neonatal and adult mice, this treatment partially restored M-AAT in the serum. Furthermore, deep sequencing confirmed both indel mutations and precise gene correction in the liver, permitting careful analysis of gene editing events in vivo. This study demonstrates a proof of concept for the application of CRISPR-Cas9 technology to correct AAT mutations in vivo and validates continued exploration of this approach for the treatment of patients with AAT deficiency.

RevDate: 2019-05-18

Rahman S, Datta M, Kim J, et al (2019)

CRISPR/Cas: An intriguing genomic editing tool with prospects in treating neurodegenerative diseases.

Seminars in cell & developmental biology pii:S1084-9521(18)30109-5 [Epub ahead of print].

The CRISPR/Cas genome editing tool has led to a revolution in biological research. Its ability to target multiple genomic loci simultaneously allows its application in gene function and genomic manipulation studies. Its involvement in the sequence specific gene editing in different backgrounds has changed the scenario of treating genetic disease. By unravelling the mysteries behind complex neuronal circuits, it not the only paved way in understanding the pathogenesis of the disease but helped in the development of large animal models of different neuronal diseases; thereby opened the gateways of successfully treating different neuronal disorders. This review explored the possibility of using of CRISPR/Cas in engineering DNA at the embryonic stage, as well as during the functioning of different cell types in the brain, to delineate implications related to the use of this super-specialized genome editing tool to overcome various neurodegenerative diseases that arise as a result of genetic mutations.

RevDate: 2019-05-17

Li M, Xie H, Liu Y, et al (2019)

Knockdown of hypoxia-inducible factor-1 alpha by tumor targeted delivery of CRISPR/Cas9 system suppressed the metastasis of pancreatic cancer.

Journal of controlled release : official journal of the Controlled Release Society pii:S0168-3659(19)30267-6 [Epub ahead of print].

The hypoxic tumor microenvironment of pancreatic cancer contributes to the progression and metastasis of tumor cells. Downregulation of hypoxia-inducible factor-1α (HIF-1α) with CRISPR/Cas9 is a promising approach to modulate tumor microenvironment and inhibit tumor metastasis. However, the in vivo delivery of CRISPR/Cas9 system remains a challenge. In the present manuscript, a tumor targeted lipid-based CRISPR/Cas9 delivery system was developed to suppress HIF-1α. Plasmids encoding Cas9 and HIF-1α-targeting sgRNA were successfully constructed and coencapsulated in R8-dGR peptide modified cationic liposome with PTX. R8-dGR-Lip exhibited enhanced BxPC-3 cell targeting and deep penetration into tumor spheroids. R8-dGR-Lip/PTX/pHIF-1α successfully downregulated HIF-1α and its downstream molecules VEGF and MMP-9, leading to enhanced antimetastatic effects. Besides, the blockade of HIF-1α also promoted the cytotoxicity of PTX on BxPC-3 cell lines. Compared with pegylated liposomes, R8-dGR-Lip enhanced the distribution in tumor tissues. The targeted delivery of CRISPR/Cas9-HIF-1α system and PTX significantly inhibited tumor growth. More importantly, inhibition of HIF-1α suppressed the metastasis of pancreatic cancer and prolonged survival time. Since CRISPR/Cas 9-HIF-1α hardly affected HIF-1α expression in normal hepatic cells, the designed R8-dGR-Lip/PTX/pHIF-1α did not induce severe toxicity in vivo. This strategy broadened the in vivo application of CRISPR/Cas9 system. Downregulation of HIF-1α may be a feasible approach for antimetastatic therapy.

RevDate: 2019-05-17

Rozov SM, EV Deineko (2019)

[Strategies for Optimizing Recombinant Protein Synthesis in Plant Cells: Classical Approaches and New Directions].

Molekuliarnaia biologiia, 53(2):179-199.

At present, pharmacologically significant proteins are synthesized in different expression systems, from bacterial to mammalian and insect cell cultures. The plant expression systems (especially suspension cell culture) combine the simplicity and low cost of bacterial systems with the ability to perform eukaryotic-type posttranslational protein modifications. A low (compared with bacterial systems) yield of the target recombinant protein is one of the shortcomings of the plant expression systems. In this review, methods, developed over the past two decades, to increase the level of recombinant gene expression and methods to prevent silencing, caused by a random insertion of the target gene into heterochromatin region, are considered. The emergence of CRISPR/Cas technologies led to the creation of a new approach to increase the gene expression level, directional insertion of "pharmaceutical" protein genes in specific, knowingly transcriptionally active genome regions. The plant cell housekeeping gene loci, actively expressed throughout the interphase, are these regions. The organization of some housekeeping genes, most promising for transferring recombinant protein genes in their loci, is considered in detail.

RevDate: 2019-05-17

Wilbie D, Walther J, E Mastrobattista (2019)

Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing.

Accounts of chemical research [Epub ahead of print].

The discovery of CRISPR/Cas has revolutionized the field of genome editing. CRIPSR/Cas components are part of the bacterial immune system and are able to induce double-strand DNA breaks in the genome, which are resolved by endogenous DNA repair mechanisms. The most relevant of these are the error-prone nonhomologous end joining and homology directed repair pathways. The former can lead to gene knockout by introduction of insertions and deletions at the cut site, while the latter can be used for gene correction based on a provided repair template. In this Account, we focus on the delivery aspects of CRISPR/Cas for therapeutic applications in vivo. Safe and effective delivery of the CRISPR/Cas components into the nucleus of affected cells is essential for therapeutic gene editing. These components can be delivered in several formats, such as pDNA, viral vectors, or ribonuclear complexes. In the ideal case, the delivery system should address the current limitations of CRISPR gene editing, which are (1) lack of targeting specific tissues or cells, (2) the inability to enter cells, (3) activation of the immune system, and (4) off-target events. To circumvent most of these problems, initial therapeutic applications of CRISPR/Cas were performed on cells ex vivo via classical methods (e.g., microinjection or electroporation) and novel methods (e.g., TRIAMF and iTOP). Ideal candidates for such methods are, for example, hematopoietic cells, but not all tissue types are suited for ex vivo manipulation. For direct in vivo application, however, delivery systems are needed that can target the CRISPR/Cas components to specific tissues or cells in the human body, without causing immune activation or causing high frequencies of off-target effects. Viral systems have been used as a first resort to transduce cells in vivo. These systems suffer from problems related to packaging constraints, immunogenicity, and longevity of Cas expression, which favors off-target events. Viral vectors are as such not the best choice for direct in vivo delivery of CRISPR/Cas. Synthetic vectors can deliver nucleic acids as well, without the innate disadvantages of viral vectors. They can be classed into lipid, polymeric, and inorganic particles, all of which have been reported in the literature. The advantage of synthetic systems is that they can deliver the CRISPR/Cas system also as a preformed ribonucleoprotein complex. The transient nature of this approach favors low frequencies of off-target events and minimizes the window of immune activation. Moreover, from a pharmaceutical perspective, synthetic delivery systems are much easier to scale up for clinical use compared to viral vectors and can be chemically functionalized with ligands to obtain target cell specificity. The first preclinical results with lipid nanoparticles delivering CRISPR/Cas either as mRNA or ribonucleoproteins are very promising. The goal is translating these CRISPR/Cas therapeutics to a clinical setting as well. Taken together, these current trends seem to favor the use of sgRNA/Cas ribonucleoprotein complexes delivered in vivo by synthetic particles.

RevDate: 2019-05-17

Luz ACO, da Silva JMA, Rezende AM, et al (2019)

Analysis of direct repeats and spacers of CRISPR/Cas systems type I-F in Brazilian clinical strains of Pseudomonas aeruginosa.

Molecular genetics and genomics : MGG pii:10.1007/s00438-019-01575-7 [Epub ahead of print].

CRISPR/Cas is an adaptive immune system found in prokaryotes, with the main function of protecting these cells from invasion and possible death by mobile genetic elements. Pseudomonas aeruginosa is considered a model for type I-F CRISPR/Cas system studies. However, its CRISPR loci characteristics have not yet been thoroughly described, and its function has not yet been fully unraveled. The aims of this study were to find the frequency of the system in Brazilian clinical isolates; to identify the loci sequence, its spacer diversity and its origins; as well as to propose a unified spacer library to aid in future structural studies of the CRISPR loci of P. aeruginosa. We investigated types I-F and I-E gene markers to establish CRISPR/Cas typing, and observed two strains harboring both systems simultaneously, a very rare feature. Through amplification and sequencing of CRISPR loci related to type I-F system, we describe polymorphisms in DRs and 350 spacers, of which 97 are new. The spacers that were identified had their possible organisms or proteins of origin identified. Spacer arrays were grouped in five different CRISPR patterns and the plasticity was inferred by rearrangements in spacer arrays. Here, we perform the first detailed and focused description of CRISPR/Cas elements in Brazilian clinical strains of P. aeruginosa. Our findings reflect active and highly diverse CRISPR loci, and we suggest that CRISPR/Cas may also pose as a transcriptional regulatory mechanism. The structural and diversity features described here can provide insights into the function of CRISPR/Cas in this pathogen and help guide the development of new therapeutic strategies.

RevDate: 2019-05-17

Pan S, Li Q, Deng L, et al (2019)

A seed motif for target RNA capture enables efficient immune defense by a Type III-B CRISPR-Cas system.

RNA biology [Epub ahead of print].

CRISPR-Cas systems provide an adaptive defense against foreign nucleic acids guided by small RNAs (crRNAs) in archaea and bacteria. The Type III CRISPR systems are reported to carry RNase, RNA-activated DNase and cyclic oligoadenylate (cOA) synthetase activity, and are significantly different from other CRISPR systems. However, detailed features of target recognition, which are essential for enhancing target specificity remain unknown in Type III CRISPR systems. Here, we show that the Type III-B Cmr-α system in S. islandicus generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3'-end of crRNA or target truncation greatly influence the target capture and cleavage by the Cmr-α effector complex. Furthermore, we found that cleavage at the tag-proximal site on the target RNA by the Cmr-α RNP complex is delayed relative to the other sites, which probably provides Cas10 more time to function as a guard against invaders. Using a mutagenesis assay in vivo, we discovered that a seed motif located at the tag-distal region of the crRNA is required by Cmr1α for target RNA capture by the Cmr-α system thereby enhancing target specificity and efficiency. These findings further refine the model for immune defense of Type III-B CRISPR-Cas system, commencing on capture, cleavage and regulation.

RevDate: 2019-05-17
CmpDate: 2019-05-17

Skakic A, Andjelkovic M, Tosic N, et al (2019)

CRISPR/Cas9 genome editing of SLC37A4 gene elucidates the role of molecular markers of endoplasmic reticulum stress and apoptosis in renal involvement in glycogen storage disease type Ib.

Gene, 703:17-25.

Glycogen storage disease type Ib (GSD Ib) is an autosomal recessive disorder, caused by a deficiency of ubiquitously expressed SLC37A4 protein. Deficiency of SLC37A4 leads to abnormal storage of glycogen in the liver and kidneys, resulting in long-term complications of renal disease and hepatocellular adenomas, whose mechanisms are poorly understood. Molecular markers of the adaptive responses to the metabolic stress caused by a deficiency of SLC37A4, such as markers related to the endoplasmic reticulum (ER) stress and unfolded protein response (UPR), have not been extensively studied. The aim of this study was to investigate the expression of molecular markers of the UPR response and apoptosis related to a deficiency of SLC37A4 in kidney cells. For that purpose, we intended to establish a human kidney cell model system for GSD Ib. The novel variant c.248G>A, found in GSD Ib patients, was introduced into the Flp-In™T-REx™-293 cell line using CRISPR/Cas9-mediated precise gene editing method, resulting in significant decrease of SLC37A4 gene expression. In this model system we used RT-qPCR analysis to investigate the expression of molecular markers of the UPR response (ATF4, DDIT3, HSPA5, and XBP1s) and apoptosis (BCL2, BAX). We demonstrated that under chronic metabolic stress conditions caused by SLC37A4 deficiency, the ER stress-induced UPR was triggered, resulting in suppression of the UPR molecular markers and cell survival promotion (decreased expression levels of ATF4, DDIT3, HSPA5, with the exception of XBP1s). However, persistent metabolic stress overrides an adaptation and induces apoptosis through increased expression of pro-apoptotic markers (decreased ratio of BCL2/BAX genes). We established a cellular model system characterized by a deficiency of SLC37A4, which presents pathological manifestations of GSD Ib in the kidney. Expression analysis in a novel model system supports the hypothesis that renal dysfunction in the GSD Ib is partly due to the ER stress and increased apoptosis.

RevDate: 2019-05-17
CmpDate: 2019-05-17

Chang SJ, Jin SC, Jiao X, et al (2019)

Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen.

PLoS pathogens, 15(4):e1007704 pii:PPATHOGENS-D-19-00088.

Typhoid toxin is a virulence factor for Salmonella Typhi and Paratyphi, the cause of typhoid fever in humans. This toxin has a unique architecture in that its pentameric B subunit, made of PltB, is linked to two enzymatic A subunits, the ADP ribosyl transferase PltA and the deoxyribonuclease CdtB. Typhoid toxin is uniquely adapted to humans, recognizing surface glycoprotein sialoglycans terminated in acetyl neuraminic acid, which are preferentially expressed by human cells. The transport pathway to its cellular targets followed by typhoid toxin after receptor binding is currently unknown. Through a genome-wide CRISPR/Cas9-mediated screen we have characterized the mechanisms by which typhoid toxin is transported within human cells. We found that typhoid toxin hijacks specific elements of the retrograde transport and endoplasmic reticulum-associated degradation machineries to reach its subcellular destination within target cells. Our study reveals unique and common features in the transport mechanisms of bacterial toxins that could serve as the bases for the development of novel anti-toxin therapeutic strategies.

RevDate: 2019-05-17
CmpDate: 2019-05-17

Ryu N, Kim MA, Choi DG, et al (2019)

CRISPR/Cas9-mediated genome editing of splicing mutation causing congenital hearing loss.

Gene, 703:83-90.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has ushered in a new era of gene therapy. In this study, we aimed to demonstrate precise CRISPR/Cas9-mediated genome editing of the splicing mutation c.919-2A > G in intron 7 of the SLC26A4 gene, which is the second most common causative gene of congenital hearing loss. We designed candidate single-guide RNAs (sgRNAs) aimed to direct the targeting of Staphylococcus aureus Cas9 to either exon 7 or exon 8 of SLC26A4. Several of the designed sgRNAs showed targeting activity, with average indel efficiencies ranging from approximately 14% to 25%. The usage of dual sgRNAs delivered both into Neuro2a cells and primary mouse embryonic fibroblasts resulted in the successful removal of large genomic fragments within the target locus. We subsequently evaluated genome editing in the presence of artificial donor templates to induce precise target modification via homology-directed repair. Using this approach, two different donor plasmids successfully introduced silent mutations within the c.919-2A region of Slc26a4 without evident off-target activities. Overall, these results indicate that CRISPR/Cas9-mediated correction of mutations in the Slc26a4 gene is a feasible therapeutic option for restoration of hearing loss.

RevDate: 2019-05-17
CmpDate: 2019-05-17

Xu WH, Liang DY, Wang Q, et al (2019)

Knockdown of KDM2A inhibits proliferation associated with TGF-β expression in HEK293T cell.

Molecular and cellular biochemistry, 456(1-2):95-104.

Lysine-specific demethylase 2A (KDM2A, also known as JHDM1A or FBXL11) plays an important role in regulating cell proliferation. However, the mechanisms on KDM2A controlling cell proliferation are varied among cell types, even controversial conclusions have been drawn. In order to elucidate the functions and underlying mechanisms for KDM2A controlling cell proliferation and apoptosis, we screened a KDM2A knockout HEK293T cell lines by CRISPR-Cas9 to illustrate the effects of KDM2A on both biological process. The results indicate that knocking down expression of KDM2A can significantly weaken HEK293T cell proliferation. The cell cycle analysis via flow cytometry demonstrate that knockdown expression of KDM2A will lead more cells arrested at G2/M phase. Through the RNA-seq analysis of the differential expressed genes between KDM2A knockdown HEK293T cells and wild type, we screened out that TGF-β pathway was significantly downregulated in KDM2A knockdown cells, which indicates that TGF-β signaling pathway might be the downstream target of KDM2A to regulate cell proliferation. When the KDM2A knockdown HEK293T cells were transient-transfected with KDM2A overexpression plasmid or treated by TGF-β agonist hydrochloride, the cell proliferation levels can be partial or completely rescued. However, the TGF-β inhibitor LY2109761 can significantly inhibit the KDM2A WT cells proliferation, but not the KDM2A knockdown HEK293T cells. Taken together, these findings suggested that KDM2A might be a key regulator of cell proliferation and cell cycle via impacting TGF-β signaling pathway.

RevDate: 2019-05-17
CmpDate: 2019-05-17

Liu W, Zhang Y, Li S, et al (2018)

Rapid and efficient CRISPR-mediated genome editing with cloning-free method.

Acta biochimica et biophysica Sinica, 50(11):1173-1175.

RevDate: 2019-05-17
CmpDate: 2019-05-17

Graham GV, Conlon JM, Abdel-Wahab YH, et al (2019)

Glucagon-like peptides-1 from phylogenetically ancient fish show potent anti-diabetic activities by acting as dual GLP1R and GCGR agonists.

Molecular and cellular endocrinology, 480:54-64.

Glucagon-like peptides-1 (GLP-1)from phylogenetically ancient fish (lamprey, dogfish, ratfish, paddlefish and bowfin) and from a teleost, the rainbow trout produced concentration-dependent stimulations of insulin release from clonal β-cells and isolated mouse islets. Lamprey and paddlefish GLP-1 were the most potent and effective. Incubation of BRIN-BD11 cells with GLP-1 receptor (GLP1R) antagonist, exendin-4 (9-39) attenuated insulinotropic activity of all peptides whereas glucagon receptor (GCGR) antagonist [des-His1,Pro4,Glu9] glucagon amide significantly decreased the activities of lamprey and paddlefish GLP-1 only. The GIP receptor antagonist GIP (6-30) Cex-K40 [Pal] attenuated the activity of bowfin GLP-1. All peptides (1 μM) produced significant increases in cAMP concentration in CHL cells transfected with GLP1R but only lamprey and paddlefish GLP-1 stimulated cAMP production in HEK293 cells transfected with GCGR. Intraperitoneal administration of lamprey and paddlefish GLP-1 (25 nmol/kg body weight) in mice produced significant decreases in blood glucose and increased insulin concentrations comparable to the effects of human GLP-1. Lamprey and paddlefish GLP-1 display potent insulinotropic activity in vitro and glucose-lowering activity in vivo that is mediated through GLP1R and GCGR so that these peptides may constitute templates for design of new antidiabetic drugs.

RevDate: 2019-05-17
CmpDate: 2019-05-17

McCormick S (2018)

Red fruit, orange fruit, orange fruit, red fruit: genome editing in tomato.

The Plant journal : for cell and molecular biology, 95(1):3-4.

RevDate: 2019-05-17
CmpDate: 2019-05-17

McNally EM (2017)

Gene Editing for the Heart: Correcting Dystrophin Mutations.

Circulation research, 121(8):896-898.

RevDate: 2019-05-16

Radovčić M, Čulo A, I Ivančić-Baće (2019)

Cas3-stimulated runaway replication of modified ColE1 plasmids in Escherichia coli is temperature dependent.

FEMS microbiology letters pii:5490330 [Epub ahead of print].

The CRISPR-Cas system constitutes an adaptive immunity system of prokaryotes against mobile genetic elements using a crRNA-mediated interference mechanism. In Type I CRISPR-Cas systems, crRNA guided by a Cascade complex recognises the matching target DNA and promotes an R-loop formation, RNA-DNA hybrid. The helicase-nuclease Cas3 protein is then recruited to the Cascade/R-loop complex where it nicks and degrades DNA. The Cas3 activity in CRISPR-Cas immunity is reduced in Δhns cells at 37°C for unknown reasons. Cas3 can also influence regulation of plasmid replication and promote uncontrolled ('runaway') replication of ColE1 plasmids independently of other CRISPR-Cas components, requiring only its helicase activity. In this work we wanted to test whether Cas3-stimulated uncontrolled plasmid replication is affected by the temperature in Δhns and/or ΔhtpG mutants. We found that Cas3-stimulated uncontrolled plasmid replication occurs only at 37°C, irrespective of the genotype of the analysed mutants, and dependent on Cas3 helicase function. We also found that plasmid replication was strongly reduced by the hns mutation at 30°C and that Cas3 could interfere with T4 phage replication at both incubation temperatures.

RevDate: 2019-05-16

Wang Y, Li X, Osmundson T, et al (2019)

Comparative Genomic Analysis of a Multidrug-Resistant Listeria monocytogenes ST477 Isolate.

Foodborne pathogens and disease [Epub ahead of print].

Listeria monocytogenes is an opportunistic human foodborne pathogen that causes severe infections with high hospitalization and fatality rates. Clonal complex 9 (CC9) contains a large number of sequence types (STs) and is one of the predominant clones distributed worldwide. However, genetic characteristics of ST477 isolates, which also belong to CC9, have never been examined, and little is known about the detail genomic traits of this food-associated clone. In this study, we sequenced and constructed the whole-genome sequence of an ST477 isolate from a frozen food sample in China and compared it with 58 previously sequenced genomes of 25 human-associated, 5 animal, and 27 food isolates consisting of 6 CC9 and 52 other clones. Phylogenetic analysis revealed that the ST477 clustered with three Canadian ST9 isolates. All phylogeny revealed that CC9 isolates involved in this study consistently possessed the invasion-related gene vip. Mobile genetic elements (MGEs), resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system were elucidated among CC9 isolates. Our ST477 isolate contained a Tn554-like transposon, carrying five arsenical-resistance genes (arsA-arsD, arsR), which was exclusively identified in the CC9 background. Compared with the ST477 genome, three Canadian ST9 isolates shared nonsynonymous nucleotide substitutions in the condensin complex gene smc and cell surface protein genes ftsA and essC. Our findings preliminarily indicate that the extraordinary success of CC9 clone in colonization of different geographical regions is likely due to conserved features harboring MGEs, functional virulence and resistance genes. ST477 and three ST9 genomes are closely related and the distinct differences between them consist primarily of changes in genes involved in multiplication and invasion, which may contribute to the prevalence of ST9 isolates in food and food processing environment.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Assimos DG (2019)

Re: CRISPR/Cas9-Mediated Glycolate Oxidase Disruption is an Efficacious and Safe Treatment for Primary Hyperoxaluria Type I.

The Journal of urology, 201(5):853-854.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Zuo E, Sun Y, Wei W, et al (2019)

Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.

Science (New York, N.Y.), 364(6437):289-292.

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Graul AI, Dulsat C, Pina P, et al (2019)

The year's new drugs and biologics 2018: Part II - News that shaped the industry in 2018.

Drugs of today (Barcelona, Spain : 1998), 55(2):131-160.

This eagle's-eye overview of the drug industry in 2018 provides insight into some of last year's top stories, including a large and still growing outbreak of Ebola in the Democratic Republic of the Congo, as well as the potential uses (and abuses) of CRISPR technology. As in previous years, we also review orphan drug development, new regulatory agency-supported programs such as Priority Medicines Scheme (PRIME) and Sakigake, pipeline attrition, and pharma/biotech mergers and acquisitions of note. Finally, we take a glimpse into the crystal ball to anticipate the new drugs that will be approved in 2019.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Xu CL, Ruan MZC, Mahajan VB, et al (2019)

Viral Delivery Systems for CRISPR.

Viruses, 11(1): pii:v11010028.

The frontiers of precision medicine have been revolutionized by the development of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 as an editing tool. CRISPR/Cas9 has been used to develop animal models, understand disease mechanisms, and validate treatment targets. In addition, it is regarded as an effective tool for genome surgery when combined with viral delivery vectors. In this article, we will explore the various viral mechanisms for delivering CRISPR/Cas9 into tissues and cells, as well as the benefits and drawbacks of each method. We will also review the history and recent development of CRISPR and viral vectors and discuss their applications as a powerful tool in furthering our exploration of disease mechanisms and therapies.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Cyranoski D, H Ledford (2018)

Genome-edited baby claim provokes international outcry.

Nature, 563(7733):607-608.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Wienert B, Martyn GE, Funnell APW, et al (2018)

Wake-up Sleepy Gene: Reactivating Fetal Globin for β-Hemoglobinopathies.

Trends in genetics : TIG, 34(12):927-940.

Disorders in hemoglobin (hemoglobinopathies) were the first monogenic diseases to be characterized and remain among the most common and best understood genetic conditions. Moreover, the study of the β-globin locus provides a textbook example of developmental gene regulation. The fetal γ-globin genes (HBG1/HBG2) are ordinarily silenced around birth, whereupon their expression is replaced by the adult β-globin genes (HBB primarily and HBD). Over 50 years ago it was recognized that mutations that cause lifelong persistence of fetal γ-globin expression ameliorate the debilitating effects of mutations in β-globin. Since then, research has focused on therapeutically reactivating the fetal γ-globin genes. Here, we summarize recent discoveries, focusing on the influence of genome editing technologies, including CRISPR-Cas9, and emerging gene therapy approaches.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Sasaki-Honda M, Jonouchi T, Arai M, et al (2018)

A patient-derived iPSC model revealed oxidative stress increases facioscapulohumeral muscular dystrophy-causative DUX4.

Human molecular genetics, 27(23):4024-4035.

Double homeobox 4 (DUX4), the causative gene of facioscapulohumeral muscular dystrophy (FSHD), is ectopically expressed in the skeletal muscle cells of FSHD patients because of chromatin relaxation at 4q35. The diminished heterochromatic state at 4q35 is caused by either large genome contractions [FSHD type 1 (FSHD1)] or mutations in genes encoding chromatin regulators, such as SMCHD1 [FSHD type 2 (FSHD2)]. However, the mechanism by which DUX4 expression is regulated remains largely unknown. Here, using a myocyte model developed from patient-derived induced pluripotent stem cells, we determined that DUX4 expression was increased by oxidative stress (OS), a common environmental stress in skeletal muscle, in both FSHD1 and FSHD2 myocytes. We generated FSHD2-derived isogenic control clones with SMCHD1 mutation corrected by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) and homologous recombination and found in the myocytes obtained from these clones that DUX4 basal expression and the OS-induced upregulation were markedly suppressed due to an increase in the heterochromatic state at 4q35. We further found that DNA damage response (DDR) was involved in OS-induced DUX4 increase and identified ataxia-telangiectasia mutated, a DDR regulator, as a mediator of this effect. Our results suggest that the relaxed chromatin state in FSHD muscle cells permits aberrant access of OS-induced DDR signaling, thus increasing DUX4 expression. These results suggest OS could represent an environmental risk factor that promotes FSHD progression.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Mans R, Wijsman M, Daran-Lapujade P, et al (2018)

A protocol for introduction of multiple genetic modifications in Saccharomyces cerevisiae using CRISPR/Cas9.

FEMS yeast research, 18(7):.

Here, two methods are described for efficient genetic modification of Saccharomyces cerevisiae using CRISPR/Cas9. The first method enables the modification of a single genetic locus using in vivo assembly of a guide RNA (gRNA) expression plasmid without the need for prior cloning. A second method using in vitro assembled plasmids that could contain up to two gRNAs was used to simultaneously introduce up to six genetic modifications (e.g. six gene deletions) in a single transformation step by transforming up to three gRNA expression plasmids simultaneously. The method is not only suitable for gene deletion but is also applicable for in vivo site-directed mutagenesis and integration of multiple DNA fragments in a single locus. In all cases, the strain transformed with the gRNA expression plasmids was equipped with a genomic integration of Spcas9, leading to strong and constitutive expression of SpCas9. The protocols detailed here have been streamlined to be executed by virtually any yeast molecular geneticist.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Carmona-Aldana F, Zampedri C, Suaste-Olmos F, et al (2018)

CTCF knockout reveals an essential role for this protein during the zebrafish development.

Mechanisms of development, 154:51-59.

Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Khanal C, McGawley EC, Overstreet C, et al (2018)

The Elusive Search for Reniform Nematode Resistance in Cotton.

Phytopathology, 108(5):532-541.

The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) has emerged as the most important plant-parasitic nematode of cotton in the United States cotton belt. Success in the development of reniform nematode-resistant upland cotton cultivars (Gossypium hirsutum L.) has not been realized despite over three decades of breeding efforts. Research approaches ranging from conventional breeding to triple species hybrids to marker-assisted selection have been employed to introgress reniform nematode resistance from other species of cotton into upland cultivars. Reniform nematode-resistant breeding lines derived from G. longicalyx were developed in 2007. However, these breeding lines displayed stunting symptoms and a hypersensitive response to reniform nematode infection. Subsequent breeding efforts focused on G. barbadense, G. aridum, G. armoreanum, and other species that have a high level of resistance to reniform nematode. Marker-assisted selection has greatly improved screening of reniform nematode-resistant lines. The use of advanced molecular techniques such as CRISPER-Cas9 systems and alternative ways such as delivery of suitable "cry" proteins and specific double-stranded RNA to nematodes will assist in developing resistant cultivars of cotton. In spite of the efforts of cotton breeders and nematologists, successes are limited only to the development of reniform nematode-resistant breeding lines. In this article, we provide an overview of the approaches employed to develop reniform nematode-resistant upland cotton cultivars in the past, progress to date, major obstacles, and some promising future research activity.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Fu Y, RM Shaw (2017)

CASAAV Technology to Examine Regulators of Heart Failure: Cause or Effect.

Circulation research, 120(12):1846-1848.

RevDate: 2019-05-16
CmpDate: 2019-05-16

Coté AJ, A Raj (2017)

FISHing Out the Details of CRISPR Genome Tracks.

Biophysical journal, 112(6):1045-1046.

RevDate: 2019-05-15

Yeo WL, Heng E, Tan LL, et al (2019)

Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes.

Biotechnology and bioengineering [Epub ahead of print].

Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes streptomycetes has enabled high efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. Novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes, enabling efficient homology directed repair (HDR)-mediated knock-in and deletion. In strains where spCas9 was non-functional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria. This article is protected by copyright. All rights reserved.

RevDate: 2019-05-15

Makarova KS, Gao L, Zhang F, et al (2019)

Unexpected connections between type VI-B CRISPR-Cas systems, bacterial natural competence, ubiquitin signaling network and DNA modification through a distinct family of membrane proteins.

FEMS microbiology letters, 366(8):.

In addition to core Cas proteins, CRISPR-Cas loci often encode ancillary proteins that modulate the activity of the respective effectors in interference. Subtype VI-B1 CRISPR-Cas systems encode the Csx27 protein that down-regulates the activity of Cas13b when the type VI-B locus is expressed in Escherichia coli. We show that Csx27 belongs to an expansive family of proteins that contain four predicted transmembrane helices and are typically encoded in predicted operons with components of the bacterial natural transformation machinery, multidomain proteins that consist of components of the ubiquitin signaling system and proteins containing the ligand-binding WYL domain and a helix-turn-helix domain. The Csx27 family proteins are predicted to form membrane channels for ssDNA that might comprise the core of a putative novel, Ub-regulated system for DNA uptake and, possibly, degradation. In addition to these associations, a distinct subfamily of the Csx27 family appears to be a part of a novel, membrane-associated system for DNA modification. In Bacteroidetes, subtype VI-B1 systems might degrade nascent transcripts of foreign DNA in conjunction with its uptake by the bacterial cell. These predictions suggest several experimental directions for the study of type VI CRISPR-Cas systems and distinct mechanisms of foreign DNA uptake and degradation in bacteria.

RevDate: 2019-05-15

Adiego-Pérez B, Randazzo P, Daran JM, et al (2019)

Multiplex Genome Editing of Microorganisms Using CRISPR-Cas.

FEMS microbiology letters pii:5489186 [Epub ahead of print].

Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing.

RevDate: 2019-05-15

Burgsdorf I, Handley KM, Bar-Shalom R, et al (2019)

Life at Home and on the Roam: Genomic Adaptions Reflect the Dual Lifestyle of an Intracellular, Facultative Symbiont.

mSystems, 4(4): pii:mSystems00057-19.

"Candidatus Synechococcus feldmannii" is a facultative intracellular symbiont of the Atlanto-Mediterranean sponge Petrosia ficiformis. Genomic information of sponge-associated cyanobacteria derives thus far from the obligate and extracellular symbiont "Candidatus Synechococcus spongiarum." Here we utilized a differential methylation-based approach for bacterial DNA enrichment combined with metagenomics to obtain the first draft genomes of "Ca. Synechococcus feldmannii." By comparative genomics, we revealed that some genomic features (e.g., iron transport mediated by siderophores, eukaryotic-like proteins, and defense mechanisms, like CRISPR-Cas [clustered regularly interspaced short palindromic repeats-associated proteins]) are unique to both symbiont types and absent or rare in the genomes of taxonomically related free-living cyanobacteria. These genomic features likely enable life under the conditions found inside the sponge host. Interestingly, there are many genomic features that are shared by "Ca. Synechococcus feldmannii" and free-living cyanobacteria, while they are absent in the obligate symbiont "Ca. Synechococcus spongiarum." These include genes related to cell surface structures, genetic regulation, and responses to environmental stress, as well as the composition of photosynthetic genes and DNA metabolism. We speculate that the presence of these genes confers on "Ca. Synechococcus feldmannii" its facultative nature (i.e., the ability to respond to a less stable environment when free-living). Our comparative analysis revealed that distinct genomic features depend on the nature of the symbiotic interaction: facultative and intracellular versus obligate and extracellular. IMPORTANCE Given the evolutionary position of sponges as one of the earliest phyla to depart from the metazoan stem lineage, studies on their distinct and exceptionally diverse microbial communities should yield a better understanding of the origin of animal-bacterium interactions. While genomes of several extracellular sponge symbionts have been published, the intracellular symbionts have, so far, been elusive. Here we compare the genomes of two unicellular cyanobacterial sponge symbionts that share an ancestor but followed different evolutionary paths-one became intracellular and the other extracellular. Counterintuitively, the intracellular cyanobacteria are facultative, while the extracellular ones are obligate. By sequencing the genomes of the intracellular cyanobacteria and comparing them to the genomes of the extracellular symbionts and related free-living cyanobacteria, we show how three different cyanobacterial lifestyles are reflected by adaptive genomic features.

RevDate: 2019-05-15

Safari F, Zare K, Negahdaripour M, et al (2019)

CRISPR Cpf1 proteins: structure, function and implications for genome editing.

Cell & bioscience, 9:36 pii:298.

CRISPR and CRISPR-associated (Cas) protein, as components of microbial adaptive immune system, allows biologists to edit genomic DNA in a precise and specific way. CRISPR-Cas systems are classified into two main classes and six types. Cpf1 is a putative type V (class II) CRISPR effector, which can be programmed with a CRISPR RNA to bind and cleave complementary DNA targets. Cpf1 has recently emerged as an alternative for Cas9, due to its distinct features such as the ability to target T-rich motifs, no need for trans-activating crRNA, inducing a staggered double-strand break and potential for both RNA processing and DNA nuclease activity. In this review, we attempt to discuss the evolutionary origins, basic architectures, and molecular mechanisms of Cpf1 family proteins, as well as crRNA designing and delivery strategies. We will also describe the novel Cpf1 variants, which have broadened the versatility and feasibility of this system in genome editing, transcription regulation, epigenetic modulation, and base editing. Finally, we will be reviewing the recent studies on utilization of Cpf1as a molecular tool for genome editing.

RevDate: 2019-05-15

McAllister KN, JA Sorg (2019)

CRISPR genome editing systems in the genus Clostridium: a timely advancement.

Journal of bacteriology pii:JB.00219-19 [Epub ahead of print].

The genus Clostridium is composed of bioproducers, which are important for the industrial production of chemicals, as well as pathogens, which are a significant burden to the patients and on the healthcare industry. Historically, even though these bacteria are well known and are commonly studied, the genetic tools to advance our understanding of these microbes have lagged behind other systems. New tools would continue the advancement of our understanding of clostridial physiology. The genetic tools available in several clostridia are not as refined as in other organisms and each exhibit their own drawbacks. With the advent of the repurposing of the CRISPR-Cas systems for genetic modification, the tools available for clostridia have improved significantly over the past four years. Several CRISPR-Cas tools, such as using wild-type Cas9, Cas9n, dCas9/CRISPRi and a newly studied Cpf1/Cas12a, are reported. These tools have the potential to greatly advance the study of clostridial species leading to future therapies or the enhanced production of industrially relevant compounds. Here we discuss the details of the CRISPR-Cas systems as well as the advances and current issues in the developed clostridial systems.

RevDate: 2019-05-15

Lee H, Dhingra Y, DG Sashital (2019)

The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.

eLife, 8: pii:44248.

CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.

RevDate: 2019-05-15
CmpDate: 2019-05-15

Jacobs CL, Badiee RK, MZ Lin (2018)

StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins.

Nature methods, 15(7):523-526.

Robust approaches for chemogenetic control of protein function would have many biological applications. We developed stabilizable polypeptide linkages (StaPLs) based on hepatitis C virus protease. StaPLs undergo autoproteolysis to cleave proteins by default, whereas protease inhibitors prevent cleavage and preserve protein function. We created StaPLs responsive to different clinically approved drugs to bidirectionally control transcription with zinc-finger-based effectors, and used StaPLs to create single-chain, drug-stabilizable variants of CRISPR-Cas9 and caspase-9.

RevDate: 2019-05-15
CmpDate: 2019-05-15

Marx V (2018)

Stem cells: lineage tracing lets single cells talk about their past.

Nature methods, 15(6):411-414.

RevDate: 2019-05-15
CmpDate: 2019-05-15

Zheng Y, Shen W, Zhang J, et al (2018)

CRISPR interference-based specific and efficient gene inactivation in the brain.

Nature neuroscience, 21(3):447-454.

CRISPR-Cas9 has been demonstrated to delete genes in postmitotic neurons. Compared to the establishment of proliferative cell lines or animal strains, it is more challenging to acquire a highly homogeneous consequence of gene editing in a stable neural network. Here we show that dCas9-based CRISPR interference (CRISPRi) can efficiently silence genes in neurons. Using a pseudotarget fishing strategy, we demonstrate that CRISPRi shows superior targeting specificity without detectable off-target activity. Furthermore, CRISPRi can achieve multiplex inactivation of genes fundamental for neurotransmitter release with high efficiency. By developing conditional CRISPRi tools targeting synaptotagmin I (Syt1), we modified the excitatory to inhibitory balance in the dentate gyrus of the mouse hippocampus and found that the dentate gyrus has distinct regulatory roles in learning and affective processes in mice. We therefore recommend CRISPRi as a useful tool for more rapid investigation of gene function in the mammalian brain.

RevDate: 2019-05-15
CmpDate: 2019-05-15

Zhou H, Liu J, Zhou C, et al (2018)

In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice.

Nature neuroscience, 21(3):440-446.

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.

RevDate: 2019-05-15
CmpDate: 2019-05-15

Wu J, Vilarino M, Suzuki K, et al (2017)

CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs.

Scientific reports, 7(1):10487.

Genome editing using programmable nucleases has revolutionized biomedical research. CRISPR-Cas9 mediated zygote genome editing enables high efficient production of knockout animals suitable for studying development and relevant human diseases. Here we report efficient disabling pancreatogenesis in pig embryos via zygotic co-delivery of Cas9 mRNA and dual sgRNAs targeting the PDX1 gene, which when combined with chimeric-competent human pluriopotent stem cells may serve as a suitable platform for the xeno-generation of human tissues and organs in pigs.

RevDate: 2019-05-14
CmpDate: 2019-05-14

Zhang R, Wu H, Z Lian (2019)

Bioinformatics analysis of evolutionary characteristics and biochemical structure of FGF5 Gene in sheep.

Gene, 702:123-132.

Fibroblast growth factor (FGF) 5 regulates the development and periodicity of hair follicles, which can affect hair traits. Loss-of-function mutations associated with long-hair phenotypes have been described in several mammalian species. Sheep is an important economic animal, however, the evolution characterizations and biological mechanism of oFGF5 (Ovis aries FGF5) gene are still poorly understood. In this study, oFGF5 gene was obtained by resequencing the whole genome of three Dorper sheep and RACE of two Kazakh sheep FGF5. We proposed FGF5 was phylogenetically related to FGF4 family and oFGF5 clearly orthologed to goat FGF5. Six loci were found from the positive selection results of FGF5 and half of them located on signal peptide. The basically similar rates of function-altering substitutions in sheep and goat lineage and the rest of the mammalian lineage of 365 SNPs indicated that the FGF5 gene was quite conservative during evolution. Homology modeling of the oFGF5 suggested that it has a highly conserved FGF superfamily domain containing 10 β-strands. Furthermore, the protein-protein docking analysis revealed that oFGF5 have the potential to form heterodimers with oFGFR1, the predicted interaction interface of FGF5-FGFR1 heterodimer was formed mainly by residues from FGF superfamily domain. Our observations suggested the evolutionary and structural biology features of oFGF5 might be relevant to its function about hair follicle development and modulating hair growth, and we confirmed our speculation by using the FGF5 gene editing sheep produced by CRISPR/Cas9 technology.

RevDate: 2019-05-14
CmpDate: 2019-05-14

Itakura Y, Inagaki S, Wada H, et al (2018)

Trynity controls epidermal barrier function and respiratory tube maturation in Drosophila by modulating apical extracellular matrix nano-patterning.

PloS one, 13(12):e0209058 pii:PONE-D-18-33522.

The outer surface of insects is covered by the cuticle, which is derived from the apical extracellular matrix (aECM). The aECM is secreted by epidermal cells during embryogenesis. The aECM exhibits large variations in structure, function, and constituent molecules, reflecting the enormous diversity in insect appearances. To investigate the molecular principles of aECM organization and function, here we studied the role of a conserved aECM protein, the ZP domain protein Trynity, in Drosophila melanogaster. We first identified trynity as an essential gene for epidermal barrier function. trynity mutation caused disintegration of the outermost envelope layer of the cuticle, resulting in small-molecule leakage and in growth and molting defects. In addition, the tracheal tubules of trynity mutants showed defects in pore-like structures of the cuticle, and the mutant tracheal cells failed to absorb luminal proteins and liquid. Our findings indicated that trynity plays essential roles in organizing nano-level structures in the envelope layer of the cuticle that both restrict molecular trafficking through the epidermis and promote the massive absorption pulse in the trachea.

RevDate: 2019-05-14
CmpDate: 2019-05-14

He YJ, Meghani K, Caron MC, et al (2018)

DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells.

Nature, 563(7732):522-526.

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.

RevDate: 2019-05-14
CmpDate: 2019-05-14

Li XF, Li SY, Dai CM, et al (2018)

PP2A inhibition by LB-100 protects retinal pigment epithelium cells from UV radiation via activation of AMPK signaling.

Biochemical and biophysical research communications, 506(1):73-80.

AMP-activated protein kinase (AMPK) signaling activation can inhibit Ultra-violet (UV) radiation (UVR)-induced retinal pigment epithelium (RPE) cell injuries. LB-100 is a novel inhibitor of protein phosphatase 2A (PP2A), the AMPKα1 phosphatase. Here, our results demonstrated that LB-100 significantly inhibited UVR-induced viability reduction, cell death and apoptosis in established ARPE-19 cells and primary murine RPE cells. LB-100 activated AMPK, nicotinamide adenine dinucleotide phosphate (NADPH) and Nrf2 (NF-E2-related factor 2) signalings, inhibiting UVR-induced oxidative injuries and DNA damage in RPE cells. Conversely, AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A mutation, almost blocked LB-100-induced RPE cytoprotection against UVR. Importantly, CRISPR/Cas9-mediated PP2A knockout mimicked and nullified LB-100-induced anti-UVR activity in RPE cells. Collectively, these results show that PP2A inhibition by LB-100 protects RPE cells from UVR via activation of AMPK signaling.

RevDate: 2019-05-14
CmpDate: 2019-05-14

Bardet M, Seeholzer T, Unterreiner A, et al (2018)

MALT1 activation by TRAF6 needs neither BCL10 nor CARD11.

Biochemical and biophysical research communications, 506(1):48-52.

The MALT1 (Mucosa associated lymphoid tissue lymphoma translocation protein 1) paracaspase couples antigen receptors on lymphocytes to downstream signaling events. Activation of MALT1 is known to involve stimulus-dependent CBM complex formation, that is, the recruitment of BCL10-bound MALT1 to a CARD-Coiled Coil protein. Beyond this canonical, CBM-dependent mechanism of MALT1 activation, recent studies suggest that MALT1 protease activity may be triggered by alternative mechanisms. For instance, the E3-ligase TRAF6 can activate MALT1 proteolytic function and induce MALT1 auto-cleavage. However, the interplay between CBM and TRAF6 with regard to MALT1 activation has remained incompletely elucidated. Here, by generating CRISPR/Cas9-derived knock-out Jurkat T-cells, we show that TRAF6 was dispensable for CARD11/BCL10-dependent MALT1 activation upon T-cell stimulation. However, ectopically-expressed TRAF6 could induce MALT1 activity in Jurkat T-cells devoid of either CARD11 or BCL10. These data provide unequivocal evidence that TRAF6-mediated MALT1 activation does not require the upstream scaffold CARD11 or the interaction between MALT1 and BCL10. Thus, TRAF6 may be part of a previously unidentified non-canonical pathway that triggers MALT1 protease activity independently of canonical CBM signalosomes.

RevDate: 2019-05-14
CmpDate: 2019-05-14

Payungwoung T, Shinzawa N, Hino A, et al (2018)

CRISPR/Cas9 system in Plasmodium falciparum using the centromere plasmid.

Parasitology international, 67(5):605-608.

The CRISPR/Cas9 nuclease system is a powerful method to genetically modify the human malarial parasite, Plasmodium falciparum. Currently, this method is carried out by co-transfection with two plasmids, one containing the Cas9 nuclease gene, and another encoding the sgRNA and the donor template DNA. However, the efficiency of modification is currently low owing to the low frequency of these plasmids in the parasites. To improve the CRISPR/Cas9 nuclease system for P. falciparum, we developed a novel method using the transgenic parasite, PfCAS9, which stably expresses the Cas9 nuclease using the centromere plasmid. To examine the efficiency of genetic modification using the PfCAS9 parasite, we performed site-directed mutagenesis of kelch13 gene, which is considered to be involved in artemisinin resistance. Our results demonstrated that the targeted mutation could be introduced with almost 100% efficiency when the transfected PfCAS9 parasites were treated with two drugs to maintain both the centromere plasmid containing the Cas9 nuclease and the plasmid having the sgRNA. Therefore, the PfCAS9 parasite is a useful parasite line for the genetic modification of P. falciparum.

RevDate: 2019-05-14
CmpDate: 2019-05-14

Remacha L, Currás-Freixes M, Torres-Ruiz R, et al (2018)

Gain-of-function mutations in DNMT3A in patients with paraganglioma.

Genetics in medicine : official journal of the American College of Medical Genetics, 20(12):1644-1651.

PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors.

METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed.

RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma.

CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.

RevDate: 2019-05-14
CmpDate: 2019-05-14

Antoniani C, Meneghini V, Lattanzi A, et al (2018)

Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human β-globin locus.

Blood, 131(17):1960-1973.

Naturally occurring, large deletions in the β-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and β-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and β-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the β-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of β-hemoglobinopathies.

RevDate: 2019-05-14
CmpDate: 2019-05-14

Gallipoli P, Giotopoulos G, Tzelepis K, et al (2018)

Glutaminolysis is a metabolic dependency in FLT3ITD acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition.

Blood, 131(15):1639-1653.

FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation-driven leukemias.

RevDate: 2019-05-14
CmpDate: 2019-05-14

Park J, Childs L, Kim D, et al (2017)

Digenome-seq web tool for profiling CRISPR specificity.

Nature methods, 14(6):548-549.

RevDate: 2019-05-13

Wilkinson M, Drabavicius G, Silanskas A, et al (2019)

Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.

Molecular cell pii:S1097-2765(19)30312-0 [Epub ahead of print].

CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Hirano S, Abudayyeh OO, Gootenberg JS, et al (2019)

Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.

Nature communications, 10(1):1968 pii:10.1038/s41467-019-09741-6.

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets bearing a protospacer adjacent motif (PAM) and complementarity to an RNA guide. Unlike other Cas9 orthologs, Corynebacterium diphtheriae Cas9 (CdCas9) recognizes the promiscuous NNRHHHY PAM. However, the CdCas9-mediated PAM recognition mechanism remains unknown. Here, we report the crystal structure of CdCas9 in complex with the guide RNA and its target DNA at 2.9 Å resolution. The structure reveals that CdCas9 recognizes the NNRHHHY PAM via a combination of van der Waals interactions and base-specific hydrogen bonds. Moreover, we find that CdCas9 exhibits robust DNA cleavage activity with the optimal 22-nucleotide length guide RNAs. Our findings highlight the mechanistic diversity of the PAM recognition by Cas9 orthologs, and provide a basis for the further engineering of the CRISPR-Cas9 genome-editor nucleases.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Herai RH (2019)

Avoiding the off-target effects of CRISPR/cas9 system is still a challenging accomplishment for genetic transformation.

Gene, 700:176-178.

The recent disclosure of a human embryo subjected to a genetic transformation using the CRISPR/cas9 system give rise to several concerns on ethical questions about its uncontrolled use in humans. Although CRISPR/cas9 has demonstrated its efficiency, this system still lacks the capability to avoid the introduction of undesirable mutations through the target genome. In this Letter, we present several undesirable impacts that CRISPR/cas9 system have in the genetic transformation of the human genome. We briefly discuss, using the very recent literature from distinct high impact journals, the main concerns related to CRISPR/cas9 to deal with off-target effects and how the research community has treated it.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Beyret E, Liao HK, Yamamoto M, et al (2019)

Single-dose CRISPR-Cas9 therapy extends lifespan of mice with Hutchinson-Gilford progeria syndrome.

Nature medicine, 25(3):419-422.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare lethal genetic disorder characterized by symptoms reminiscent of accelerated aging. The major underlying genetic cause is a substitution mutation in the gene coding for lamin A, causing the production of a toxic isoform called progerin. Here we show that reduction of lamin A/progerin by a single-dose systemic administration of adeno-associated virus-delivered CRISPR-Cas9 components suppresses HGPS in a mouse model.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Santiago-Fernández O, Osorio FG, Quesada V, et al (2019)

Development of a CRISPR/Cas9-based therapy for Hutchinson-Gilford progeria syndrome.

Nature medicine, 25(3):423-426.

CRISPR/Cas9-based therapies hold considerable promise for the treatment of genetic diseases. Among these, Hutchinson-Gilford progeria syndrome, caused by a point mutation in the LMNA gene, stands out as a potential candidate. Here, we explore the efficacy of a CRISPR/Cas9-based approach that reverts several alterations in Hutchinson-Gilford progeria syndrome cells and mice by introducing frameshift mutations in the LMNA gene.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Nelson CE, Wu Y, Gemberling MP, et al (2019)

Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy.

Nature medicine, 25(3):427-432.

Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy1-6, however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice7; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Hsu JY, Fulco CP, Cole MA, et al (2018)

CRISPR-SURF: discovering regulatory elements by deconvolution of CRISPR tiling screen data.

Nature methods, 15(12):992-993.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Ikeda K, Uchida N, Nishimura T, et al (2018)

Efficient scarless genome editing in human pluripotent stem cells.

Nature methods, 15(12):1045-1047.

Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Rusk N (2018)

Recording transcriptional activity.

Nature methods, 15(12):999.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Callaway E (2018)

Ban on 'gene drives' is back on the UN's agenda - worrying scientists.

Nature, 563(7732):454-455.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Ma H, Tu LC, Naseri A, et al (2018)

CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging.

Nature methods, 15(11):928-931.

Clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA scaffolds have been adapted to carry multiple binding sites for fluorescent proteins to enhance brightness for live cell imaging of genomic loci. However, many of these modifications result in guide RNA instability and thus produce lower genome-labeling efficiency than anticipated. Here we introduce CRISPR-Sirius, based on octet arrays of aptamers conferring both enhanced guide RNA stability and brightness, and provide initial biological applications of this platform.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Bubeck F, Hoffmann MD, Harteveld Z, et al (2018)

Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9.

Nature methods, 15(11):924-927.

Anti-CRISPR proteins are powerful tools for CRISPR-Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR-Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Rusk N (2018)

Keeping CRISPR in check.

Nature methods, 15(11):857.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Ting PY, Parker AE, Lee JS, et al (2018)

Guide Swap enables genome-scale pooled CRISPR-Cas9 screening in human primary cells.

Nature methods, 15(11):941-946.

CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Tang L (2018)

Does the number of chromosomes matter?.

Nature methods, 15(10):761.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Willi M, Smith HE, Wang C, et al (2018)

Mutation frequency is not increased in CRISPR-Cas9-edited mice.

Nature methods, 15(10):756-758.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Marx V (2018)

Base editing a CRISPR way.

Nature methods, 15(10):767-770.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Wang Q, Chung CY, Rosenberg JN, et al (2018)

Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

Methods in molecular biology (Clifton, N.J.), 1850:237-257.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of fucose significantly enhances antibody dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) to disrupt the α-1,6-fucosyltranferase (FUT8) gene and subsequently inhibit α-1,6-fucosylation on antibodies expressed in CHO cells.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Kellner K, Solanki A, Amann T, et al (2018)

Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells.

Methods in molecular biology (Clifton, N.J.), 1850:221-235.

MicroRNAs with their unique ability to target hundreds of genes have been highlighted as powerful tools to improve bioprocess behavior of cells. The common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, which requires the introduction and expression of extra genetic material. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate Chinese hamster ovary (CHO) cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in controlling CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We showed that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures making it a potentially interesting target to improve bioprocess performance of CHO cells.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Parola C, Mason DM, Zingg A, et al (2018)

Genome Engineering of Hybridomas to Generate Stable Cell Lines for Antibody Expression.

Methods in molecular biology (Clifton, N.J.), 1850:79-111.

From the perspective of academic and small research laboratories, the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells, resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression, however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization. In order to bridge the gap between the simplicity of transient transfection and consistent production by stable cell lines, we describe a method to stably integrate antibody genes into the endogenous immunogenomic loci of hybridoma cells using CRISPR/Cas9 genome editing. Initially, the antibody variable light (VL) chain is deleted by multiplexed Cas9 cleavage; subsequently, the variable heavy (VH) chain is replaced by a fluorescent reporter gene (mRuby) by Cas9-assisted homology-directed repair (HDR). This cell line is customized by replacing mRuby with a synthetic antibody (consisting of a VL, light constant region and VH) by once again using Cas9-assisted HDR. Due to hybridomas' inherent ability to surface display and secrete antibodies, they provide a suitable host for both the selection and the production process, substantially streamlining the process for stable cell line generation, and thus we refer to this platform as plug-and-(dis)play (PnP) hybridomas.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Roychoudhury P, De Silva Feelixge H, Reeves D, et al (2018)

Viral diversity is an obligate consideration in CRISPR/Cas9 designs for targeting the HIV reservoir.

BMC biology, 16(1):75.

BACKGROUND: RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative approach for HIV. However, most studies to date have focused on molecular clones with ideal target site recognition and do not account for target site variability observed within and between patients. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV.

RESULTS: We identified a set of gRNAs targeting HIV LTR, gag, and pol using publicly available sequences for these genes and ranked gRNAs according to global conservation across HIV-1 group M and within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all globally available sequences. We then selected 59 gRNAs from our list of highly conserved LTR target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-infected patients, we found that globally conserved sites also had greater within-host target conservation. Lastly, we developed a mathematical model based on varying distributions of within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the number of doses required to deplete the latent reservoir and achieve functional cure thresholds. Our modeling results highlight the importance of within-host target site conservation. While increased doses may overcome low target cleavage efficiency, inadequate targeting of rare strains is predicted to lead to rebound upon cART cessation even with many doses.

CONCLUSIONS: Target site selection must account for global and within host viral genetic diversity. Globally conserved target sites are good starting points for design, but multiplexing is essential for depleting quasispecies and preventing viral load rebound upon therapy cessation.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Codner GF, Mianné J, Caulder A, et al (2018)

Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.

BMC biology, 16(1):70.

BACKGROUND: Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method.

RESULTS: We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors.

CONCLUSION: lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Lanza DG, Gaspero A, Lorenzo I, et al (2018)

Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles.

BMC biology, 16(1):69.

BACKGROUND: The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs).

RESULTS: Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected.

CONCLUSIONS: Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Takahashi M, T Hirota (2018)

Dynamics of sister chromatids through the cell cycle: Together and apart.

The Journal of cell biology, 217(6):1887-1889.

When and how sister chromatid resolution occurs after DNA replication is a fundamental question. Stanyte et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201801157) used CRISPR/Cas9 technology to label and track genomic loci in live cells throughout the cell cycle, shedding light on how replication is linked to mitotic sister chromatid organization.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Schleussner N, Merkel O, Costanza M, et al (2018)

The AP-1-BATF and -BATF3 module is essential for growth, survival and TH17/ILC3 skewing of anaplastic large cell lymphoma.

Leukemia, 32(9):1994-2007.

Transcription factor AP-1 is constitutively activated and IRF4 drives growth and survival in ALK+ and ALK- anaplastic large cell lymphoma (ALCL). Here we demonstrate high-level BATF and BATF3 expression in ALCL. Both BATFs bind classical AP-1 motifs and interact with in ALCL deregulated AP-1 factors. Together with IRF4, they co-occupy AP-1-IRF composite elements, differentiating ALCL from non-ALCL. Gene-specific inactivation of BATFs, or global AP-1 inhibition results in ALCL growth retardation and/or cell death in vitro and in vivo. Furthermore, the AP-1-BATF module establishes TH17/group 3 innate lymphoid cells (ILC3)-associated gene expression in ALCL cells, including marker genes such as AHR, IL17F, IL22, IL26, IL23R and RORγt. Elevated IL-17A and IL-17F levels were detected in a subset of children and adolescents with ALK+ ALCL. Furthermore, a comprehensive analysis of primary lymphoma data confirms TH17-, and in particular ILC3-skewing in ALCL compared with PTCL. Finally, pharmacological inhibition of RORC as single treatment leads to cell death in ALCL cell lines and, in combination with the ALK inhibitor crizotinib, enforces death induction in ALK+ ALCL. Our data highlight the crucial role of AP-1/BATFs in ALCL and lead to the concept that some ALCL might originate from ILC3.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Nabet B, Roberts JM, Buckley DL, et al (2018)

The dTAG system for immediate and target-specific protein degradation.

Nature chemical biology, 14(5):431-441.

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

RevDate: 2019-05-13
CmpDate: 2019-05-13

Cooper ML, Choi J, Staser K, et al (2018)

An "off-the-shelf" fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies.

Leukemia, 32(9):1970-1983.

T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide-resistant, allo-tolerant "off-the-shelf" CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphoma without a requirement for autologous T cells.

RevDate: 2019-05-12

Bruch R, Urban GA, C Dincer (2019)

CRISPR/Cas Powered Multiplexed Biosensing.

Multiplexed CRISPR/Cas biosensing offers various possibilities for diagnosing different diseases by analyzing many analytes from one single specimen. However, in this relatively new field, nearly no multiplexing approaches exist, as many challenges need to be addressed. We discuss the reasons behind it and possible strategies to push the multiplexing level.

RevDate: 2019-05-11

Trasanidou D, Gerós AS, Mohanraju P, et al (2019)

Keeping crispr in check: diverse mechanisms of phage-encoded anti-crisprs.

FEMS microbiology letters pii:5488435 [Epub ahead of print].

CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools.

RevDate: 2019-05-11

Wang L, J Li (2019)

"Artificial spermatid"-mediated genome editing.

Biology of reproduction pii:5488429 [Epub ahead of print].

For years, extensive efforts have been made to use mammalian sperm as the mediator to generate genetically modified animals, however the strategy of sperm-mediated gene transfer (SMGT) is unable to produce stable and diversified modifications in descendants. Recently, haploid embryonic stem cells (haESCs) have been successfully derived from haploid embryos carrying the genome of highly specialized gametes, and can stably maintain haploidy (through periodic cell sorting based on DNA quantity) and both self-renewal and pluripotency in long-term cell culture. In particular, haESCs derived from androgenetic haploid blastocysts (AG-haESCs), carrying only the sperm genome, can support the generation of live mice (semi-cloned, SC mice) through oocyte injection. Remarkably, after removal of the imprinted control regions H19-DMR (differentially methylated region of DNA) and IG-DMR in AG-haESCs, the double knockout (DKO)-AG-haESCs can stably produce SC animals with high efficiency, and so can serve as a sperm equivalent. Importantly, DKO-AG-haESCs can be used for multiple rounds of gene modifications in vitro, followed by efficient generation of live and fertile mice with the expected genetic traits. Thus, DKO-AG-haESCs (referred to as "artificial spermatids") combed with CRISPR-Cas technology can be used as the genetically tractable fertilization agent to efficiently create genetically-modified offspring, and is a versatile genetic tool for in vivo analyses of gene function.

RevDate: 2019-05-11

Cañez C, Selle K, Goh YJ, et al (2019)

Outcomes and characterization of Chromosomal Self-Targeting by Native CRISPR-Cas Systems in Streptococcus thermophilus.

FEMS microbiology letters pii:5488433 [Epub ahead of print].

CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair bacterial pathways, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4-400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems.

RevDate: 2019-05-11

Nasef M, Muffly MC, Beckman AB, et al (2019)

Regulation of cyclic oligoadenylate synthesis by the S. epidermidis Cas10-Csm complex.

RNA (New York, N.Y.) pii:rna.070417.119 [Epub ahead of print].

CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg2+ dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.

RevDate: 2019-05-10

Farhat S, Jain N, Singh N, et al (2019)

CRISPR-cas 9 directed genome engineering for enhancing salt stress tolerance in rice.

Seminars in cell & developmental biology pii:S1084-9521(18)30119-8 [Epub ahead of print].

Crop productivity in rice is harshly limited due to high concentration of salt in the soil. To understand the intricacies of the mechanism it is important to unravel the key pathways operating inside the plant cell. Emerging state-of-the art technologies have provided the tools to discover the key components inside the plant cell for salt tolerance. Among the molecular entities, transcription factors and/or other important components of sensing and signaling cascades have been the attractive targets and the role of NHX and SOS1 transporters amply described. Not only marker assisted programs but also transgenic approaches by using reverse genetic strategies (knockout or knockdown) or overexpression have been extensively used to engineer rice crop. CRISPR/Cas is an attractive paradigm and provides the feasibility for manipulating several genes simultaneously. Here, in this review we highlight some of the molecular entities that could be potentially targeted for generating rice amenable to sustain growth under high salinity conditions by employing CRISPR/Cas. We also try to address key questions for rice salt stress tolerance other than what is already known.

RevDate: 2019-05-10

Schmöhl F, Peters V, Schmitt CP, et al (2019)

CNDP1 knockout in zebrafish alters the amino acid metabolism, restrains weight gain, but does not protect from diabetic complications.

Cellular and molecular life sciences : CMLS pii:10.1007/s00018-019-03127-z [Epub ahead of print].

The gene CNDP1 was associated with the development of diabetic nephropathy. Its enzyme carnosinase 1 (CN1) primarily hydrolyzes the histidine-containing dipeptide carnosine but other organ and metabolic functions are mainly unknown. In our study we generated CNDP1 knockout zebrafish, which showed strongly decreased CN1 activity and increased intracellular carnosine levels. Vasculature and kidneys of CNDP1-/- zebrafish were not affected, except for a transient glomerular alteration. Amino acid profiling showed a decrease of certain amino acids in CNDP1-/- zebrafish, suggesting a specific function for CN1 in the amino acid metabolisms. Indeed, we identified a CN1 activity for Ala-His and Ser-His. Under diabetic conditions increased carnosine levels in CNDP1-/- embryos could not protect from respective organ alterations. Although, weight gain through overfeeding was restrained by CNDP1 loss. Together, zebrafish exhibits CN1 functions, while CNDP1 knockout alters the amino acid metabolism, attenuates weight gain but cannot protect organs from diabetic complications.

RevDate: 2019-05-10

Wang C, Jiang S, Ke L, et al (2019)

Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma.

The Journal of biological chemistry pii:RA119.008793 [Epub ahead of print].

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients poorly respond to treatment. New treatments are therefore needed to improve outcomes of NPC. To better understand the molecular pathogenesis of NPC, here we used NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways associated with NPC (i.e. dependency factors). This screen identified the MYST histone acetyl transferase complex; NF-kB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene (MDM2) as NPC dependency factors/pathways. Using cell growth, cDNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines, but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.

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ESP Quick Facts

ESP Origins

In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

ESP Support

In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

ESP Rationale

Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

ESP Goal

In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

ESP Usage

Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

ESP Content

When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

ESP Help

Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

ESP Plans

With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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CRISPR-Cas

By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

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Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin (and even a collection of poetry — Chicago Poems by Carl Sandburg).

Timelines

ESP now offers a much improved and expanded collection of timelines, designed to give the user choice over subject matter and dates.

Biographies

Biographical information about many key scientists.

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are now being automatically maintained and generated on the ESP site.

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