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Bibliography on: CRISPR-Cas

The Electronic Scholarly Publishing Project: Providing world-wide, free access to classic scientific papers and other scholarly materials, since 1993.


ESP: PubMed Auto Bibliography 01 Apr 2020 at 01:36 Created: 


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: 2020-03-31

Pyenson NC, LA Marraffini (2020)

Co-evolution within structured bacterial communities results in multiple expansion of CRISPR loci and enhanced immunity.

eLife, 9: pii:53078.

Type II CRISPR-Cas systems provide immunity against phages and plasmids that infect bacteria through the insertion of a short sequence from the invader's genome, known as the 'spacer', into the CRISPR locus. Spacers are transcribed into guide RNAs that direct the Cas9 nuclease to its target on the invader. In liquid cultures, most bacteria acquire a single spacer. Multiple spacer integration is a rare event which significance for immunity is poorly understood. Here, we found that when phage infections occur on solid media, a high proportion of the surviving colonies display complex morphologies that contain cells with multiple spacers. This is the result of the viral-host co-evolution, in which the immunity provided by the initial acquired spacer is easily overcome by escaper phages. Our results reveal the versatility of CRISPR-Cas immunity, which can respond with both single or multiple spacer acquisition schemes to solve challenges presented by different environments.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Yamazaki H, Shirakawa K, Matsumoto T, et al (2020)

APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression.

PloS one, 15(1):e0223463.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Becú-Villalobos D (2019)

[CRISPR-CAS9 in medicine, the saga continues].

Medicina, 79(6):522-523.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Straiton J (2019)

Genetically modified humans: the X-Men of scientific research.

BioTechniques, 66(6):249-252.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Campenhout CV, Cabochette P, Veillard AC, et al (2019)

Guidelines for optimized gene knockout using CRISPR/Cas9.

BioTechniques, 66(6):295-302.

CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Yang B, Yang L, J Chen (2019)

Development and Application of Base Editors.

The CRISPR journal, 2(2):91-104.

Base editing is emerging as a potent new strategy to achieve precise gene editing. By combining different nucleobase deaminases with Cas9 or Cpf1 proteins, several base editors have recently been developed to achieve targeted base conversions in different genomic contexts. Importantly, base editors have been successfully applied in animals, plants, and bacteria to induce precise substitutions at the single-base level with high efficiency. In this review, we summarize recent progress in the development and application of base editors and discuss some of the future directions of the technology.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Borowicz P, Chan H, Medina D, et al (2020)

A simple and efficient workflow for generation of knock-in mutations in Jurkat T cells using CRISPR/Cas9.

Scandinavian journal of immunology, 91(4):e12862.

CRISPR/Cas9 is a powerful gene-editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9-mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock-in mutations were introduced in Jurkat T cells by homologous directed repair using single-stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus-dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele-specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock-in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Guo CJ, Allen BM, Hiam KJ, et al (2019)

Depletion of microbiome-derived molecules in the host using Clostridium genetics.

Science (New York, N.Y.), 366(6471):.

The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp., the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes-derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A-modulatory activity.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Yarra R, Jin L, Zhao Z, et al (2019)

Progress in Tissue Culture and Genetic Transformation of Oil Palm: An Overview.

International journal of molecular sciences, 20(21):.

Oil palm (Elaeis guineensis, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands of palm oil consumption. However, tissue culture and biotechnological approaches can resolve these concerns. Over the past three decades, significant research has been carried out to develop tissue culture and genetic transformation protocols for oil palm. Somatic embryogenesis is an efficient platform for the micropropagation of oil palm on a large scale. In addition, various genetic transformation techniques, including microprojectile bombardment, Agrobacterium tumefaciens mediated, Polyethylene glycol mediated mediated, and DNA microinjection, have been developed by optimizing various parameters for the efficient genetic transformation of oil palm. This review mainly emphasizes the methods established for in vitro propagation and genetic transformation of oil palm. Finally, we propose the application of the genome editing tool CRISPR/Cas9 to improve the various traits in this oil yielding crop.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Ermert AL, Nogué F, Stahl F, et al (2019)

CRISPR/Cas9-Mediated Knockout of Physcomitrella patens Phytochromes.

Methods in molecular biology (Clifton, N.J.), 2026:237-263.

Here we describe procedures for gene disruption and excision in Physcomitrella using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated 9) methods, exemplarily targeting phytochrome (PHY) gene loci. Thereby double-strand breaks (DSBs) are induced using a single guide RNA (sgRNA) with the Cas9 nuclease, leading to insertions or deletions (indels) due to incorrect repair by the nonhomologous-end joining (NHEJ) mechanism. We also include protocols for excision of smaller genomic fragments or whole genes either with or without homologous recombination-assisted repair. The protocol can be adapted to target several loci simultaneously, thereby allowing the physiological analysis of phenotypes that would be masked by functional redundancy. In our particular case, multiple PHY gene knockouts would likely be valuable in understanding phytochrome functions in mosses and, perhaps, higher plants too. Target sites for site-directed induction of DSBs are predicted with the CRISPOR online-tool and are inserted in silico into sequence matrices for the design of sgRNA expression cassettes. The resulting DNAs are cloned into Gateway DONOR vectors and the respective expression plasmids used for moss cotransformation with a Cas9 expression plasmid and a selectable marker (either on a separate plasmid or on one of the other plasmids). After the selection process, genomic DNA is extracted and transformants are analyzed by PCR fingerprinting.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Ng CL, DA Fidock (2019)

Plasmodium falciparum In Vitro Drug Resistance Selections and Gene Editing.

Methods in molecular biology (Clifton, N.J.), 2013:123-140.

Malaria continues to be a global health burden, threatening over 40% of the world's population. Drug resistance in Plasmodium falciparum, the etiological agent of the majority of human malaria cases, is compromising elimination efforts. New approaches to treating drug-resistant malaria benefit from defining resistance liabilities of known antimalarial agents and compounds in development and defining genetic changes that mediate loss of parasite susceptibility. Here, we present protocols for in vitro selection of drug-resistant parasites and for site-directed gene editing of candidate resistance mediators to test for causality.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Hua K, Zhang J, Botella JR, et al (2019)

Perspectives on the Application of Genome-Editing Technologies in Crop Breeding.

Molecular plant, 12(8):1047-1059.

Most conventional and modern crop-improvement methods exploit natural or artificially induced genetic variations and require laborious characterization of the progenies of multiple generations derived from time-consuming genetic crosses. Genome-editing systems, in contrast, provide the means to rapidly modify genomes in a precise and predictable way, making it possible to introduce improvements directly into elite varieties. Here, we describe the range of applications available to agricultural researchers using existing genome-editing tools. In addition to providing examples of genome-editing applications in crop breeding, we discuss the technical and social challenges faced by breeders using genome-editing tools for crop improvement.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Wong WR, Brugman KI, Maher S, et al (2019)

Autism-associated missense genetic variants impact locomotion and neurodevelopment in Caenorhabditis elegans.

Human molecular genetics, 28(13):2271-2281.

Autism spectrum disorder (ASD) involves thousands of alleles in over 850 genes, but the current functional inference tools are not sufficient to predict phenotypic changes. As a result, the causal relationship of most of these genetic variants in the pathogenesis of ASD has not yet been demonstrated and an experimental method prioritizing missense alleles for further intensive analysis is crucial. For this purpose, we have designed a pipeline that uses Caenorhabditis elegans as a genetic model to screen for phenotype-changing missense alleles inferred from human ASD studies. We identified highly conserved human ASD-associated missense variants in their C. elegans orthologs, used a CRISPR/Cas9-mediated homology-directed knock-in strategy to generate missense mutants and analyzed their impact on behaviors and development via several broad-spectrum assays. All tested missense alleles were predicted to perturb protein function, but we found only 70% of them showed detectable phenotypic changes in morphology, locomotion or fecundity. Our findings indicate that certain missense variants in the C. elegans orthologs of human CACNA1D, CHD7, CHD8, CUL3, DLG4, GLRA2, NAA15, PTEN, SYNGAP1 and TPH2 impact neurodevelopment and movement functions, elevating these genes as candidates for future study into ASD. Our approach will help prioritize functionally important missense variants for detailed studies in vertebrate models and human cells.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Sheng Z, Wang X, Ma Y, et al (2019)

MS-based strategies for identification of protein SUMOylation modification.

Electrophoresis, 40(21):2877-2887.

Protein SUMOylation modification conjugated with small ubiquitin-like modifiers (SUMOs) is one kind of PTMs, which exerts comprehensive roles in cellular functions, including gene expression regulation, DNA repair, intracellular transport, stress responses, and tumorigenesis. With the development of the peptide enrichment approaches and MS technology, more than 6000 SUMOylated proteins and about 40 000 SUMO acceptor sites have been identified. In this review, we summarize several popular approaches that have been developed for the identification of SUMOylated proteins in human cells, and further compare their technical advantages and disadvantages. And we also introduce identification approaches of target proteins which are co-modified by both SUMOylation and ubiquitylation. We highlight the emerging trends in the SUMOylation field as well. Especially, the advent of the clustered regularly interspaced short palindromic repeats/ Cas9 technique will facilitate the development of MS for SUMOylation identification.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Darbellay F, Bochaton C, Lopez-Delisle L, et al (2019)

The constrained architecture of mammalian Hox gene clusters.

Proceedings of the National Academy of Sciences of the United States of America, 116(27):13424-13433.

In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, as the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters toward a tighter structure during the evolution of the vertebrate lineage. Here, we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster, such as to invert one or several transcription units, with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in cis.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Ciurkot K, Vonk B, Gorochowski TE, et al (2019)

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art.

Journal of visualized experiments : JoVE.

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S. cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Cheong KH, Koh JM, MC Jones (2019)

Black Swans of CRISPR: Stochasticity and Complexity of Genetic Regulation.

BioEssays : news and reviews in molecular, cellular and developmental biology, 41(7):e1900032.

Recent waves of controversies surrounding genetic engineering have spilled into popular science in Twitter battles between reputable scientists and their followers. Here, a cautionary perspective on the possible blind spots and risks of CRISPR and related biotechnologies is presented, focusing in particular on the stochastic nature of cellular control processes.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Han X, Wang M, Duan S, et al (2019)

Generation of hypoimmunogenic human pluripotent stem cells.

Proceedings of the National Academy of Sciences of the United States of America, 116(21):10441-10446.

Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage "don't-eat me" signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Noble C, Min J, Olejarz J, et al (2019)

Daisy-chain gene drives for the alteration of local populations.

Proceedings of the National Academy of Sciences of the United States of America, 116(17):8275-8282.

If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. "Daisy-drive" systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Enderle J, Dorn A, Beying N, et al (2019)

The Protease WSS1A, the Endonuclease MUS81, and the Phosphodiesterase TDP1 Are Involved in Independent Pathways of DNA-protein Crosslink Repair in Plants.

The Plant cell, 31(4):775-790.

DNA-protein crosslinks (DPCs) represent a severe threat to the genome integrity; however, the main mechanisms of DPC repair were only recently elucidated in humans and yeast. Here we define the pathways for DPC repair in plants. Using CRISPR/Cas9, we could show that only one of two homologs of the universal repair proteases SPARTAN/ weak suppressor of smt3 (Wss1), WSS1A, is essential for DPC repair in Arabidopsis (Arabidopsis thaliana). WSS1A defective lines exhibit developmental defects and are hypersensitive to camptothecin (CPT) and cis-platin. Interestingly, the CRISPR/Cas9 mutants of TYROSYL-DNA PHOSPHODIESTERASE 1 (TDP1) are insensitive to CPT, and only the wss1A tdp1 double mutant reveals a higher sensitivity than the wss1A single mutant. This indicates that TDP1 defines a minor backup pathway in the repair of DPCs. Moreover, we found that knock out of the endonuclease METHYL METHANESULFONATE AND UV SENSITIVE PROTEIN 81 (MUS81) results in a strong sensitivity to DPC-inducing agents. The fact that wss1A mus81 and tdp1 mus81 double mutants exhibit growth defects and an increase in dead cells in root meristems after CPT treatment demonstrates that there are three independent pathways for DPC repair in Arabidopsis. These pathways are defined by their different biochemical specificities, as main actors, the DNA endonuclease MUS81 and the protease WSS1A, and the phosphodiesterase TDP1 as backup.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Zhu W, Saw D, Weiss M, et al (2019)

Induction of Brain Arteriovenous Malformation Through CRISPR/Cas9-Mediated Somatic Alk1 Gene Mutations in Adult Mice.

Translational stroke research, 10(5):557-565.

Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Khadempar S, Familghadakchi S, Motlagh RA, et al (2019)

CRISPR-Cas9 in genome editing: Its function and medical applications.

Journal of cellular physiology, 234(5):5751-5761.

The targeted genome modification using RNA-guided nucleases is associated with several advantages such as a rapid, easy, and efficient method that not only provides the manipulation and alteration of genes and functional studies for researchers, but also increases their awareness of the molecular basis of the disease and development of new and targeted therapeutic approaches. Different techniques have been emerged so far as the molecular scissors mediating targeted genome editing including zinc finger nuclease, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). CRISPR-Cas9 is a bacterial immune system against viruses in which the single-strand RNA-guided Cas9 nuclease is linked to the targeted complementary sequences to apply changes. The advances made in the transfer, modification, and emergence of specific solutions have led to the creation of different classes of CRISPR-Cas9. Since this robust tool is capable of direct correction of disease-causing mutations, its ability to treat genetic disorders has attracted the tremendous attention of researchers. Considering the reported cases of nonspecific targeting of Cas9 proteins, many studies focused on enhancing the Cas9 features. In this regard, significant advances have been made in choosing guide RNA, new enzymes and methods for identifying misplaced targeting. Here, we highlighted the history and various direct aspects of CRISPR-Cas9, such as precision in genomic targeting, system transfer and its control over correction events with its applications in future biological studies, and modern treatment of diseases.

RevDate: 2020-03-29

Wu R, Chai B, Cole JR, et al (2020)

Targeted assemblies of cas1 suggest CRISPR-Cas's response to soil warming.

The ISME journal pii:10.1038/s41396-020-0635-1 [Epub ahead of print].

There is an increasing interest in the clustered regularly interspaced short palindromic repeats CRISPR-associated protein (CRISPR-Cas) system to reveal potential virus-host dynamics. The universal and most conserved Cas protein, cas1 is an ideal marker to elucidate CRISPR-Cas ecology. We constructed eight Hidden Markov Models (HMMs) and assembled cas1 directly from metagenomes by a targeted-gene assembler, Xander, to improve detection capacity and resolve the diverse CRISPR-Cas systems. The eight HMMs were first validated by recovering all 17 cas1 subtypes from the simulated metagenome generated from 91 prokaryotic genomes across 11 phyla. We challenged the targeted method with 48 metagenomes from a tallgrass prairie in Central Oklahoma recovering 3394 cas1. Among those, 88 were near full length, 5 times more than in de-novo assemblies from the Oklahoma metagenomes. To validate the host assignment by cas1, the targeted-assembled cas1 was mapped to the de-novo assembled contigs. All the phylum assignments of those mapped contigs were assigned independent of CRISPR-Cas genes on the same contigs and consistent with the host taxonomies predicted by the mapped cas1. We then investigated whether 8 years of soil warming altered cas1 prevalence within the communities. A shift in microbial abundances was observed during the year with the biggest temperature differential (mean 4.16 °C above ambient). cas1 prevalence increased and even in the phyla with decreased microbial abundances over the next 3 years, suggesting increasing virus-host interactions in response to soil warming. This targeted method provides an alternative means to effectively mine cas1 from metagenomes and uncover the host communities.

RevDate: 2020-03-29

Garcia-Doval C, Schwede F, Berk C, et al (2020)

Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6.

Nature communications, 11(1):1596 pii:10.1038/s41467-020-15334-5.

Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.

RevDate: 2020-03-28

George AF, Jang KS, Nyegaard M, et al (2020)

Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling.

Human reproduction (Oxford, England) pii:5812617 [Epub ahead of print].

STUDY QUESTION: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)?

SUMMARY ANSWER: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization.

WHAT IS KNOWN ALREADY: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis.

STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization.

eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs.

SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization.

LARGE SCALE DATA: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640.

This study is limited to in vitro analyses.

Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation.

This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.

RevDate: 2020-03-28

Mahendra C, Christie KA, Osuna BA, et al (2020)

Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer.

Nature microbiology, 5(4):620-629.

CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9-Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR-Cas9 inhibition.

RevDate: 2020-03-28

Walton RT, Christie KA, Whittaker MN, et al (2020)

Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.

Science (New York, N.Y.) pii:science.aba8853 [Epub ahead of print].

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG capable of targeting an expanded set of NGN PAMs, and further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN>NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

RevDate: 2020-03-28

Tang LC, F Gu (2020)

Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM.

Yi chuan = Hereditas, 42(3):236-249.

The emergence of the gene editing technology, especial CRISPR-Cas (clustered regularly intersected short palindromic repeats and CRISPR associated proteins), has greatly promoted the ability of human beings to transform natural species. It has been widely harnessed for the engineering in the medical, industrial, agricultural and other fields. The key component of the CRISPR-Cas system, the Cas protein, possesses its specific features, including self-activity, recognition site, cutting end and guide RNA. PAM (protein assistant motif) is a number of nucleotides adjacent to the target site, which is very important for the Cas protein to recognize the target sequence and is also the key characteristic of CRISPR-Cas. There are several reported methods for identification of PAM. In this review, we summarize the searching for the Cas protein, the identification of Cas mutants with desired traits and the mapping of the PAM (including the extending of PAM spectrum), in order to provide a reference for the development and optimization of next-generation gene editing tools.

RevDate: 2020-03-27

Karunarathna NL, Wang H, Harloff HJ, et al (2020)

Elevating seed oil content in a polypoid crop by induced mutations in SEED FATTY ACID REDUCER genes.

Plant biotechnology journal [Epub ahead of print].

Plant based oils are valuable agricultural products, and seed oil content (SOC) is the major yield component in oil crops. Increasing SOC has been successfully targeted through the selection and genetic modification of oil biosynthesis. The SOC in rapeseed declined during the seed maturation and eventually caused the final accumulated seed oil quantity. However, genes involved in oil degradation during seed maturity are not deeply studied so far. We performed a candidate gene association study using a world-wide collection of rapeseed germplasm. We identified SEED FATTY ACID REDUCER (SFAR) genes, which had a significant effect on SOC and fatty acid (FA) composition. SFAR genes belong to the GDSL lipases, and GDSL lipases have a broad range of functions in plants. After quantification of gene expression using RNA-seq and quantitative PCR, we used targeted (CRISPR-Cas mediated) and random (chemical) mutagenesis to modify turnover rates of seed oil in winter rapeseed. For the first time, we demonstrate significant increase of SOC in a crop after knocking out members of the BnSFAR4 and BnSFAR5 gene families without pleiotropic effects on seed germination, vigor, and oil mobilization. Our results offer new perspectives for improving oil yield by targeted mutagenesis.

RevDate: 2020-03-26

Meyer M, Kuffner K, Winter J, et al (2020)

Myocyte Enhancer Factor 2A (MEF2A) Defines Oxytocin-Induced Morphological Effects and Regulates Mitochondrial Function in Neurons.

International journal of molecular sciences, 21(6): pii:ijms21062200.

The neuropeptide oxytocin (OT) is a well-described modulator of socio-emotional traits, such as anxiety, stress, social behavior, and pair bonding. However, when dysregulated, it is associated with adverse psychiatric traits, such as various aspects of autism spectrum disorder (ASD). In this study, we identify the transcription factor myocyte enhancer factor 2A (MEF2A) as the common link between OT and cellular changes symptomatic for ASD, encompassing neuronal morphology, connectivity, and mitochondrial function. We provide evidence for MEF2A as the decisive factor defining the cellular response to OT: while OT induces neurite retraction in MEF2A expressing neurons, OT causes neurite outgrowth in absence of MEF2A. A CRISPR-Cas-mediated knockout of MEF2A and retransfection of an active version or permanently inactive mutant, respectively, validated our findings. We also identified the phosphatase calcineurin as the main upstream regulator of OT-induced MEF2A signaling. Further, MEF2A signaling dampens mitochondrial functioning in neurons, as MEF2A knockout cells show increased maximal cellular respiration, spare respiratory capacity, and total cellular ATP. In summary, we reveal a central role for OT-induced MEF2A activity as major regulator of cellular morphology as well as neuronal connectivity and mitochondrial functioning, with broad implications for a potential treatment of disorders based on morphological alterations or mitochondrial dysfunction.

RevDate: 2020-03-26
CmpDate: 2020-03-26

Matson AW, Hosny N, Swanson ZA, et al (2019)

Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system.

PloS one, 14(12):e0226107.

The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.

RevDate: 2020-03-26
CmpDate: 2020-03-26

Elso CM, Scott NA, Mariana L, et al (2019)

Replacing murine insulin 1 with human insulin protects NOD mice from diabetes.

PloS one, 14(12):e0225021.

Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (Ins) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.

RevDate: 2020-03-26
CmpDate: 2020-03-26

Liu X, Zhang P, Zhang X, et al (2020)

Fgf21 knockout mice generated using CRISPR/Cas9 reveal genetic alterations that may affect hair growth.

Gene, 733:144242.

OBJECTIVE: To investigate fibroblast growth factor 21 (Fgf21) alterations that may affect hair growth and the underlying molecular mechanisms by constructing Fgf21 global knockout (KO) mice using microinjection-mediated CRISPR/Cas9.

RESULTS: Following genomic DNA sequencing, we identified 18 mice carrying Ffg21 mutations among the total 63 offspring mice obtained by injecting 340 embryos, which yielded a mutation rate of 28.6 percent. Of these 18 mice, three had both alleles knocked out and 15 were monoallelic KO mice. Compared with the wild-type (WT) mice, the phenotypic analysis showed that the litter size of Fgf21 KO mice significantly reduced (p < 0.05), but physiological indexes of the birth weight, gender rate, body weight (0-8 week) and body weight of adult male and female were no significant difference (p > 0.05). Compared to WT mice, physiological anatomy indicated that the morphological characters of vital organs in Fgf21 KO mice were normal. Depilation experiments demonstrated that compared to the WT mice, the hair regrowth speed was reduced in the Fgf21 KO mice. The number of hair shafts in these mice considerably decreased, as indicated by the tissue sample analyses. Real-time quantitative PCR showed that Erk and Akt expression in the KO mice was significantly decreased (P < 0.05), whereas western blotting demonstrated that the expression of Erk and Akt proteins and their phosphorylation levels in KO mice decreased at different rates (P < 0.05).

CONCLUSIONS: Fgf21 was shown to affect hair follicle development and growth cycle, which may be associated with Pi3k/Akt and Mapk/Erk signaling pathways.

RevDate: 2020-03-26
CmpDate: 2020-03-26

Kaiser J (2019)

CRISPR reveals some cancer drugs hit unexpected targets.

Science (New York, N.Y.), 365(6458):1065.

RevDate: 2020-03-27
CmpDate: 2020-03-27

Wang H, Nakamura M, Abbott TR, et al (2019)

CRISPR-mediated live imaging of genome editing and transcription.

Science (New York, N.Y.), 365(6459):1301-1305.

We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.

RevDate: 2020-03-27
CmpDate: 2020-03-27

Wang D, Stoveken HM, Zucca S, et al (2019)

Genetic behavioral screen identifies an orphan anti-opioid system.

Science (New York, N.Y.), 365(6459):1267-1273.

Opioids target the μ-opioid receptor (MOR) to produce unrivaled pain management, but their addictive properties can lead to severe abuse. We developed a whole-animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in Caenorhabditis elegans, we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR, and inhibits signaling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of C. elegans as a scalable platform for genetic discovery of G protein-coupled receptor signaling principles.

RevDate: 2020-03-26
CmpDate: 2020-03-26

Davis TH (2019)

Profile of Rodolphe Barrangou.

Proceedings of the National Academy of Sciences of the United States of America, 116(32):15754-15756.

RevDate: 2020-03-27
CmpDate: 2020-03-27

Ali Z, Zaidi SS, Tashkandi M, et al (2019)

A Simplified Method to Engineer CRISPR/Cas9-Mediated Geminivirus Resistance in Plants.

Methods in molecular biology (Clifton, N.J.), 2028:167-183.

Throughout the world, geminiviruses cause devastating losses in economically important crops, including tomato, cotton, cassava, potato, chili, and cucumber; however, control mechanisms such as genetic resistance remain expensive and ineffective. CRISPR/Cas9 is an adaptive immunity mechanism used by prokaryotes to defend against invading nucleic acids of phages and plasmids. The CRISPR/Cas9 system has been harnessed for targeted genome editing in a variety of eukaryotic species, and in plants, CRISPR/Cas9 has been used to modify or introduce many traits, including virus resistance. Recently, we demonstrated that the CRISPR/Cas9 system could be used to engineer plant immunity against geminiviruses by directly targeting the viral genome for degradation. In this chapter, we describe a detailed method for engineering CRISPR/Cas9-mediated resistance against geminiviruses. This method may provide broad, durable viral resistance, as it can target conserved regions of the viral genome and can also be customized to emerging viral variants. Moreover, this method can be used in many crop species, as it requires little or no knowledge of the host plant's genome.

RevDate: 2020-03-27
CmpDate: 2020-03-27

Osmani Z, Jin S, Mikami M, et al (2019)

CRISPR/Cas9-Mediated Editing of Genes Encoding rgs-CaM-like Proteins in Transgenic Potato Plants.

Methods in molecular biology (Clifton, N.J.), 2028:153-165.

A tobacco calmodulin-like protein, rgs-CaM, has been shown to interact with viruses in a variety of ways; it contributes to geminivirus infections but is also involved in primed immunity to the cucumber mosaic virus. Sequence similarity searches revealed several calmodulin-like proteins similar to rgs-CaM (rCML) in Arabidopsis and other Solanaceae plants, including potato (Solanum tuberosum). To analyze the functions of each rCML, mutations were introduced into potato rCMLs using the CRISPR/Cas9 system. Here, we describe our protocol of the CRISPR/Cas9-mediated targeted mutagenesis in stably transformed potato plants.

RevDate: 2020-03-26
CmpDate: 2020-03-26

Baffoe-Bonnie MS (2019)

A justice-based argument for including sickle cell disease in CRISPR/Cas9 clinical research.

Bioethics, 33(6):661-668.

CRISPR/Cas9 is quickly becoming one of the most influential biotechnologies of the last five years. Clinical trials will soon be underway to test whether CRISPR/Cas9 can edit away the genetic mutations that cause sickle cell disease (SCD). This article will present the background of CRISPR/Cas9 gene editing and SCD, highlighting research that supports the application of CRISPR/Cas9 to SCD. While much has been written on why SCD is a good biological candidate for CRISPR/Cas9, less has been written on the ethical implications of including SCD in CRISPR/Cas9 research. This article will argue that there is a strong case in favor of including SCD. Three benefits are achieving distributive justice in research, continuing to repair the negative relationship between patients with SCD and the health-care system, and benefit-sharing for those who do not directly participate in CRISPR/Cas9 research. Opponents will argue that SCD is a risky candidate, that researchers will not find willing participants, and that the burden of SCD is low. Of this set of arguments, the first gives pause. However, on balance, the case in favor of including SCD in CRISPR/Cas9 research is stronger than the case against. Ultimately, this article will show that the historic and sociopolitical injustices that impede progress in treating and curing SCD can be alleviated through biotechnology.

RevDate: 2020-03-27
CmpDate: 2020-03-27

Zhong Z, Sretenovic S, Ren Q, et al (2019)

Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG.

Molecular plant, 12(7):1027-1036.

Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBEC1 and PmCDA1 cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDA1, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

RevDate: 2020-03-27
CmpDate: 2020-03-27

Hua K, Tao X, Han P, et al (2019)

Genome Engineering in Rice Using Cas9 Variants that Recognize NG PAM Sequences.

Molecular plant, 12(7):1003-1014.

CRISPR/Cas9 genome editing relies on sgRNA-target DNA base pairing and a short downstream PAM sequence to recognize target DNA. The strict protospacer adjacent motif (PAM) requirement hinders applications of the CRISPR/Cas9 system since it restricts the targetable sites in the genomes. xCas9 and SpCas9-NG are two recently engineered SpCas9 variants that can recognize more relaxed NG PAMs, implying a great potential in addressing the issue of PAM constraint. Here we use stable transgenic lines to evaluate the efficacies of xCas9 and SpCas9-NG in performing gene editing and base editing in rice. We found that xCas9 can efficiently induce mutations at target sites with NG and GAT PAM sequences in rice. However, base editors containing xCas9 failed to edit most of the tested target sites. SpCas9-NG exhibited a robust editing activity at sites with various NG PAMs without showing any preference for the third nucleotide after NG. Moreover, we showed that xCas9 and SpCas9-NG have higher specificity than SpCas9 at the CGG PAM site. We further demonstrated that different forms of cytosine or adenine base editors containing SpCas9-NG worked efficiently in rice with broadened PAM compatibility. Taken together, our work has yielded versatile genome-engineering tools that will significantly expand the target scope in rice and other crops.

RevDate: 2020-03-27
CmpDate: 2020-03-27

Ren B, Liu L, Li S, et al (2019)

Cas9-NG Greatly Expands the Targeting Scope of the Genome-Editing Toolkit by Recognizing NG and Other Atypical PAMs in Rice.

Molecular plant, 12(7):1015-1026.

CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM), such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate OsBZR1 gain-of-function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants.

RevDate: 2020-03-27
CmpDate: 2020-03-27

Porter SN, Levine RM, SM Pruett-Miller (2019)

A Practical Guide to Genome Editing Using Targeted Nuclease Technologies.

Comprehensive Physiology, 9(2):665-714.

Genome engineering using programmable nucleases is a rapidly evolving technique that enables precise genetic manipulations within complex genomes. Although this technology first surfaced with the creation of meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases, CRISPR-Cas9 has been the most widely adopted platform because of its ease of use. This comprehensive review presents a basic overview of genome engineering and discusses the major technological advances in the field. In addition to nucleases, we discuss CRISPR-derived base editors and epigenetic modifiers. We also delve into practical applications of these tools, including creating custom-edited cell and animal models as well as performing genetic screens. Finally, we discuss the potential for therapeutic applications and ethical considerations related to employing this technology in humans. © 2019 American Physiological Society. Compr Physiol 9:665-714, 2019.

RevDate: 2020-03-26
CmpDate: 2020-03-26

Oser MG, Fonseca R, Chakraborty AA, et al (2019)

Cells Lacking the RB1 Tumor Suppressor Gene Are Hyperdependent on Aurora B Kinase for Survival.

Cancer discovery, 9(2):230-247.

Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating RB1 and TP53 mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the RB1 gene product RB1 is to repress the E2F transcription factor family. RB1 also plays both E2F-dependent and E2F-independent mitotic roles. We performed a synthetic lethal CRISPR/Cas9 screen in an RB1-/- SCLC cell line that conditionally expresses RB1 to identify dependencies that are caused by RB1 loss and discovered that RB1-/- SCLC cell lines are hyperdependent on multiple proteins linked to chromosomal segregation, including Aurora B kinase. Moreover, we show that an Aurora B kinase inhibitor is efficacious in multiple preclinical SCLC models at concentrations that are well tolerated in mice. These results suggest that RB1 loss is a predictive biomarker for sensitivity to Aurora B kinase inhibitors in SCLC and perhaps other RB1-/- cancers. SIGNIFICANCE: SCLC is rarely associated with actionable protooncogene mutations. We did a CRISPR/Cas9-based screen that showed that RB1-/- SCLC are hyperdependent on AURKB, likely because both genes control mitotic fidelity, and confirmed that Aurora B kinase inhibitors are efficacious against RB1-/- SCLC tumors in mice at nontoxic doses.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.

RevDate: 2020-03-25

Buglo E, Sarmiento E, Martuscelli NB, et al (2020)

Genetic compensation in a stable slc25a46 mutant zebrafish: A case for using F0 CRISPR mutagenesis to study phenotypes caused by inherited disease.

PloS one, 15(3):e0230566 pii:PONE-D-19-22413.

A phenomenon of genetic compensation is commonly observed when an organism with a disease-bearing mutation shows incomplete penetrance of the disease phenotype. Such incomplete phenotypic penetrance, or genetic compensation, is more commonly found in stable knockout models, rather than transient knockdown models. As such, these incidents present a challenge for the disease modeling field, although a deeper understanding of genetic compensation may also hold the key for novel therapeutic interventions. In our study we created a knockout model of slc25a46 gene, which is a recently discovered important player in mitochondrial dynamics, and deleterious mutations in which are known to cause peripheral neuropathy, optic atrophy and cerebellar ataxia. We report a case of genetic compensation in a stable slc25a46 homozygous zebrafish mutant (hereafter referred as "mutant"), in contrast to a penetrant disease phenotype in the first generation (F0) slc25a46 mosaic mutant (hereafter referred as "crispant"), generated with CRISPR/Cas-9 technology. We show that the crispant phenotype is specific and rescuable. By performing mRNA sequencing, we define significant changes in slc25a46 mutant's gene expression profile, which are largely absent in crispants. We find that among the most significantly altered mRNAs, anxa6 gene stands out as a functionally relevant player in mitochondrial dynamics. We also find that our genetic compensation case does not arise from mechanisms driven by mutant mRNA decay. Our study contributes to the growing evidence of the genetic compensation phenomenon and presents novel insights about Slc25a46 function. Furthermore, our study provides the evidence for the efficiency of F0 CRISPR screens for disease candidate genes, which may be used to advance the field of functional genetics.

RevDate: 2020-03-25
CmpDate: 2020-03-25

Yu Z, Lv H, Wu Y, et al (2019)

Enhancement of FK520 production in Streptomyces hygroscopicus by combining traditional mutagenesis with metabolic engineering.

Applied microbiology and biotechnology, 103(23-24):9593-9606.

FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production.

RevDate: 2020-03-25
CmpDate: 2020-03-25

Dong Z, Long J, Huang L, et al (2019)

Construction and application of an HSP70 promoter-inducible genome editing system in transgenic silkworm to induce resistance to Nosema bombycis.

Applied microbiology and biotechnology, 103(23-24):9583-9592.

The microsporidian Nosema bombycis is an obligate intracellular parasitic fungus that causes devastating disease in sericulture. To date, no efficient biotechnological method to inhibit the proliferation of microspores has been established. Here, we developed a powerful genetic engineering technique involving microsporidia-inducible genome editing in transgenic silkworm that confers resistance to N. bombycis. This system includes an HSP70 promoter-induced expression of the Cas9 protein line and a target BmATAD3A gene line. The double-positive HSP70-Cas9(+)×sgATAD3A(+) lines were obtained by hybridization and activation of the CRISPR/Cas9 system under the condition of microsporidia infection, although it is silenced in uninfected individuals. Genome editing analysis showed that the system could efficiently edit the BmATAD3A gene and induce large deletions. It is notable that the HSP70-induced system could effectively improve the survival rate of transgenic silkworm after microsporidia infection and inhibit the expression of key microsporidia genes. Moreover, no significant developmental differences between the transgenic silkworms infected with microsporidia and normal individuals were observed. In this study, we effectively inhibited microsporidia proliferation in transgenic individuals through disruptive techniques, thereby providing a method for microsporidia treatment and prevention, paving the way for economically advantageous insect breeding.

RevDate: 2020-03-25
CmpDate: 2020-03-25

Orvedahl A, McAllaster MR, Sansone A, et al (2019)

Autophagy genes in myeloid cells counteract IFNγ-induced TNF-mediated cell death and fatal TNF-induced shock.

Proceedings of the National Academy of Sciences of the United States of America, 116(33):16497-16506.

Host inflammatory responses must be tightly regulated to ensure effective immunity while limiting tissue injury. IFN gamma (IFNγ) primes macrophages to mount robust inflammatory responses. However, IFNγ also induces cell death, and the pathways that regulate IFNγ-induced cell death are incompletely understood. Using genome-wide CRISPR/Cas9 screening, we identified autophagy genes as central mediators of myeloid cell survival during the IFNγ response. Hypersensitivity of autophagy gene-deficient cells to IFNγ was mediated by tumor necrosis factor (TNF) signaling via receptor interacting protein kinase 1 (RIPK1)- and caspase 8-mediated cell death. Mice with myeloid cell-specific autophagy gene deficiency exhibited marked hypersensitivity to fatal systemic TNF administration. This increased mortality in myeloid autophagy gene-deficient mice required the IFNγ receptor, and mortality was completely reversed by pharmacologic inhibition of RIPK1 kinase activity. These findings provide insight into the mechanism of IFNγ-induced cell death via TNF, demonstrate a critical function of autophagy genes in promoting cell viability in the presence of inflammatory cytokines, and implicate this cell survival function in protection against mortality during the systemic inflammatory response.

RevDate: 2020-03-25
CmpDate: 2020-03-25

Cohen J (2019)

CRISPR patent fight revived.

Science (New York, N.Y.), 365(6448):15-16.

RevDate: 2020-03-25
CmpDate: 2020-03-25

Zhang D, Hurst T, Duan D, et al (2019)

Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design.

Proceedings of the National Academy of Sciences of the United States of America, 116(18):8693-8698.

While CRISPR/Cas9 is a powerful tool in genome engineering, the on-target activity and off-target effects of the system widely vary because of the differences in guide RNA (gRNA) sequences and genomic environments. Traditional approaches rely on separate models and parameters to treat on- and off-target cleavage activities. Here, we demonstrate that a free-energy scheme dominates the Cas9 editing efficacy and delineate a method that simultaneously considers on-target activities and off-target effects. While data-driven machine-learning approaches learn rules to model particular datasets, they may not be as transferrable to new systems or capable of producing new mechanistic insights as principled physical approaches. By integrating the energetics of R-loop formation under Cas9 binding, the effect of the protospacer adjacent motif sequence, and the folding stability of the whole single guide RNA, we devised a unified, physical model that can apply to any cleavage-activity dataset. This unified framework improves predictions for both on-target activities and off-target efficiencies of spCas9 and may be readily transferred to other systems with different guide RNAs or Cas9 ortholog proteins.

RevDate: 2020-03-24
CmpDate: 2020-03-24

Li B, Clohisey SM, Chia BS, et al (2020)

Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection.

Nature communications, 11(1):164.

Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.

RevDate: 2020-03-24
CmpDate: 2020-03-24

Wang SR, Wu LY, Huang HY, et al (2020)

Conditional control of RNA-guided nucleic acid cleavage and gene editing.

Nature communications, 11(1):91.

Prokaryotes use repetitive genomic elements termed CRISPR (clustered regularly interspaced short palindromic repeats) to destroy invading genetic molecules. Although CRISPR systems have been widely used in DNA and RNA technology, certain adverse effects do occur. For example, constitutively active CRISPR systems may lead to a certain risk of off-target effects. Here, we introduce post-synthetic masking and chemical activation of guide RNA (gRNA) to controlling CRISPR systems. An RNA structure profiling probe (2-azidomethylnicotinic acid imidazolide) is used. Moreover, we accomplish conditional control of gene editing in live cells. This proof-of-concept study demonstrates promising potential of chemical activation of gRNAs as a versatile tool for chemical biology.

RevDate: 2020-03-24
CmpDate: 2020-03-24

Katzmeier F, Aufinger L, Dupin A, et al (2019)

A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a.

PloS one, 14(12):e0220091.

Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of ≈ 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.

RevDate: 2020-03-24
CmpDate: 2020-03-24

Kotagama OW, Jayasinghe CD, T Abeysinghe (2019)

Era of Genomic Medicine: A Narrative Review on CRISPR Technology as a Potential Therapeutic Tool for Human Diseases.

BioMed research international, 2019:1369682.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) provides acquired immunity in microorganisms against exogenous DNA that may hinder the survival of the organism. Pioneering work by Doudna and Charpentier in 2012 resulted in the creation of the CRISPR/Cas9 genome editing tool on the basis of this concept. The aim of this was to create a rapid, efficient, and versatile genome-editing tool to facilitate genetic manipulation. The mechanism relies on two components: the RNA guide which acts as a sentinel and a Cas protein complex which functions as a highly precise molecular knife. The guide RNA can be modified to match a DNA sequence of interest in the cell and accordingly be used to rectify mutations that may otherwise cause disease. Within a few years following the development of the CRISPR/Cas9 tool, its usage has become ubiquitous. Its influence extends into many fields of biological sciences from biotechnology and biochemistry to molecular biology and biomedical sciences. The following review aims at shedding some light on to the applications of the CRISPR/Cas9 tool in the field of biomedical sciences, particularly gene therapy. An insight with relation to a few of the many diseases that are being tackled with the aid of the CRISPR/Cas9 mechanism and the trends, successes, and challenges of this application as a gene therapy are discussed in this review.

RevDate: 2020-03-24
CmpDate: 2020-03-24

Fandino RA, Haverkamp A, Bisch-Knaden S, et al (2019)

Mutagenesis of odorant coreceptor Orco fully disrupts foraging but not oviposition behaviors in the hawkmoth Manduca sexta.

Proceedings of the National Academy of Sciences of the United States of America, 116(31):15677-15685.

The hawkmoth Manduca sexta and one of its preferred hosts in the North American Southwest, Datura wrightii, share a model insect-plant relationship based on mutualistic and antagonistic life-history traits. D. wrightii is the innately preferred nectar source and oviposition host for M. sexta Hence, the hawkmoth is an important pollinator while the M. sexta larvae are specialized herbivores of the plant. Olfactory detection of plant volatiles plays a crucial role in the behavior of the hawkmoth. In vivo, the odorant receptor coreceptor (Orco) is an obligatory component for the function of odorant receptors (ORs), a major receptor family involved in insect olfaction. We used CRISPR-Cas9 targeted mutagenesis to knock out (KO) the MsexOrco gene to test the consequences of a loss of OR-mediated olfaction in an insect-plant relationship. Neurophysiological characterization revealed severely reduced antennal and antennal lobe responses to representative odorants emitted by D. wrightii In a wind-tunnel setting with a flowering plant, Orco KO hawkmoths showed disrupted flight orientation and an ablated proboscis extension response to the natural stimulus. The Orco KO gravid female displayed reduced attraction toward a nonflowering plant. However, more than half of hawkmoths were able to use characteristic odor-directed flight orientation and oviposit on the host plant. Overall, OR-mediated olfaction is essential for foraging and pollination behaviors, but plant-seeking and oviposition behaviors are sustained through additional OR-independent sensory cues.

RevDate: 2020-03-24
CmpDate: 2020-03-24

Takeda H, Kataoka S, Nakayama M, et al (2019)

CRISPR-Cas9-mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes.

Proceedings of the National Academy of Sciences of the United States of America, 116(31):15635-15644.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-β (TGF-β) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.

RevDate: 2020-03-23

Borges AL, Castro B, Govindarajan S, et al (2020)

Bacterial alginate regulators and phage homologs repress CRISPR-Cas immunity.

Nature microbiology pii:10.1038/s41564-020-0691-3 [Epub ahead of print].

CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection1. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases2. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB-AlgB), previously characterized in the regulation of Pseudomonas aeruginosa alginate biosynthesis3,4, as a regulator of the expression and activity of the P. aeruginosa Type I-F CRISPR-Cas system. Downstream of KinB-AlgB, activators of alginate production AlgU (a σE orthologue) and AlgR repress CRISPR-Cas activity during planktonic and surface-associated growth5. AmrZ, another alginate regulator6, is triggered to repress CRISPR-Cas immunity upon surface association. Pseudomonas phages and plasmids have taken advantage of this regulatory scheme and carry hijacked homologs of AmrZ that repress CRISPR-Cas expression and activity. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.

RevDate: 2020-03-23

Marino ND, Pinilla-Redondo R, Csörgő B, et al (2020)

Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies.

Nature methods pii:10.1038/s41592-020-0771-6 [Epub ahead of print].

Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, a diverse family of prokaryotic adaptive immune systems, have emerged as a biotechnological tool and therapeutic. The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, enables the development of more controllable and precise CRISPR-Cas tools. Here we discuss applications of Acr proteins for post-translational control of CRISPR-Cas systems in prokaryotic and mammalian cells, organisms and ecosystems.

RevDate: 2020-03-23

Otten ABC, BK Sun (2020)

Research Techniques Made Simple: CRISPR Genetic Screens.

The Journal of investigative dermatology, 140(4):723-728.e1.

CRISPR and Cas proteins, often referred to as CRISPR/Cas, are the components of a bacterial genome editing system that can be used to perturb genes in cells and tissues. A classic application is to use CRISPR/Cas to generate genetic loss-of-function. When performed at large scale and combined with deep sequencing techniques, CRISPR-based perturbations can be performed in a high throughput setting to screen many candidate genomic elements for their roles in a phenotype of interest. Here, we discuss major considerations in the design, execution, and analysis of CRISPR screens. We focus on CRISPR knockout screens but also review adaptations to the CRISPR/Cas system that highlight the versatility of the system to make other types of experimental genetic changes as well. We also discuss examples of CRISPR genetic screens in investigative dermatology and how they may be used to answer key scientific questions in the field.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Davis BJ, MR O'Connell (2020)

Put on Your Para-spectacles: The Development of Optimized CRISPR-Cas13-Based Approaches to Image RNA Dynamics in Real Time.

Molecular cell, 77(2):207-209.

Live-cell RNA imaging is a powerful approach to observe the real-time dynamics of RNA metabolism. Two recent papers describe an optimized fluorescence-based CRISPR-Cas13 approach to image colocalized or repeat-containing RNAs in real time, as well as demonstrate simultaneous RNA-DNA labeling by using Cas13 and Cas9 in tandem.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Wang C, Wang G, Gao Y, et al (2020)

A cytokinin-activation enzyme-like gene improves grain yield under various field conditions in rice.

Plant molecular biology, 102(4-5):373-388.

KEY MESSAGE: CRISPR-edited variants at the 3'-end of OsLOGL5's coding sequence (CDS), significantly increased rice grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Cytokinins impact numerous aspects of plant growth and development. This study reports that constitutive ectopic overexpression of a rice cytokinin-activation enzyme-like gene, OsLOGL5, significantly reduced primary root growth, tiller number, and yield. Conversely, mutations at the 3'-end of OsLOGL5 CDS resulted in normal rice plant morphology but with increased grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Six gene edited variants (Edit A to F) were created and tested in the field. Edit-B and Edit-F plants increased, but Edit-D and Edit-E plants decreased, the panicle number per plant. All OsLOGL5-edited plants significantly increased seed setting rate, total grain numbers, full-filled grain numbers per panicle, and thousand seed weight under drought conditions, suggesting that OsLOGL5 is likely involved in the regulation of both seed development and grain filling processes. Our results indicate that the C-terminal end of OsLOGL5 protein plays an important role in regulating rice yield improvement under different abiotic stress conditions, and OsLOGL5 is important for rice yield enhancement and stability.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Shimokawa-Chiba N, Müller C, Fujiwara K, et al (2019)

Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis.

Nature communications, 10(1):5397.

Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Karponi G, Kritas SK, Papadopoulou G, et al (2019)

Development of a CRISPR/Cas9 system against ruminant animal brucellosis.

BMC veterinary research, 15(1):422.

BACKGROUND: Brucellosis, caused by several Brucella species, such as the bacterium Brucella melitensis, is considered one of the most severe zoonotic diseases worldwide. Not only does it affect ruminant animal populations, leading to a substantial financial burden for stockbreeders, but also poses severe public health issues. For almost four decades in southern Europe and elsewhere, eradication of the disease has been based on ambiguously effective programs, rendering massive sanitation of livestock urgent and indispensable. Gene therapy, which has been proved effective in the clinic, could possibly constitute an alternative option towards a permanent cure for brucellosis, by aiding in the deletion or inactivation of genes associated with the replication of Brucella within the host cells.

RESULTS: We infected ovine macrophages with B.melitensis, to simulate the host cell/microorganism interaction in vitro, and transduced the infected cells with CRISPR/Cas9 lentiviral vectors that target Brucella's RNA polymerase subunit A (RpolA) or virulence-associated gene virB10 at a multiplicity of infection of 60. We demonstrate a significant decrease in the bacterial load per cell when infected cells are transduced with the RpolA vector and that the number of internalized brucellae per cell remains unaffected when macrophages are transduced with a conventional lentiviral vector expressing the green fluorescence protein, thus underlining the bactericidal effect of our CRISPR/Cas9 system.

CONCLUSIONS: Pending in vivo verification of our findings, overall, these results may prove critical not only for the treatment of human brucellosis, but for other infectious diseases in general.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Shridhar PB, Worley JN, Gao X, et al (2019)

Analysis of virulence potential of Escherichia coli O145 isolated from cattle feces and hide samples based on whole genome sequencing.

PloS one, 14(11):e0225057.

Escherichia coli O145 serogroup is one of the big six non-O157 Shiga toxin producing E. coli (STEC) that causes foodborne illnesses in the United States and other countries. Cattle are a major reservoir of STEC, which harbor them in their hindgut and shed in the feces. Cattle feces is the main source of hide and subsequent carcass contaminations during harvest leading to foodborne illnesses in humans. The objective of our study was to determine the virulence potential of STEC O145 strains isolated from cattle feces and hide samples. A total of 71 STEC O145 strains isolated from cattle feces (n = 16), hide (n = 53), and human clinical samples (n = 2) were used in the study. The strains were subjected to whole genome sequencing using Illumina MiSeq platform. The average draft genome size of the fecal, hide, and human clinical strains were 5.41, 5.28, and 5.29 Mb, respectively. The average number of genes associated with mobile genetic elements was 260, 238, and 259, in cattle fecal, hide, and human clinical strains, respectively. All strains belonged to O145:H28 serotype and carried eae subtype γ. Shiga toxin 1a was the most common Shiga toxin gene subtype among the strains, followed by stx2a and stx2c. The strains also carried genes encoding type III secretory system proteins, nle, and plasmid-encoded virulence genes. Phylogenetic analysis revealed clustering of cattle fecal strains separately from hide strains, and the human clinical strains were more closely related to the hide strains. All the strains belonged to sequence type (ST)-32. The virulence gene profile of STEC O145 strains isolated from cattle sources was similar to that of human clinical strains and were phylogenetically closely related to human clinical strains. The genetic analysis suggests the potential of cattle STEC O145 strains to cause human illnesses. Inclusion of more strains from cattle and their environment in the analysis will help in further elucidation of the genetic diversity and virulence potential of cattle O145 strains.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Peng Q, Fang L, Ding Z, et al (2020)

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology.

Journal of virological methods, 276:113772.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-ΔORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-ΔORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Cheng W, Wang S, Zhang Z, et al (2019)

CRISPR-Cas9 Screens Identify the RNA Helicase DDX3X as a Repressor of C9ORF72 (GGGGCC)n Repeat-Associated Non-AUG Translation.

Neuron, 104(5):885-898.e8.

Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells. We found that DDX3X, an RNA helicase, suppresses the repeat-associated non-AUG translation of GGGGCC repeats. DDX3X directly binds to (GGGGCC)n RNAs but not antisense (CCCCGG)n RNAs. Its helicase activity is essential for the translation repression. Reduction of DDX3X increases DPR levels in C9ORF72-ALS/FTD patient cells and enhances (GGGGCC)n-mediated toxicity in Drosophila. Elevating DDX3X expression is sufficient to decrease DPR levels, rescue nucleocytoplasmic transport abnormalities, and improve survival of patient iPSC-differentiated neurons. This work identifies genetic modifiers of DPR protein production and provides potential therapeutic targets for C9ORF72-ALS/FTD.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Gordon DM, Adeosun SO, Ngwudike SI, et al (2019)

CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation.

Archives of biochemistry and biophysics, 672:108072.

Obesity is the predominant cause of non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance and diabetes. NAFLD includes a spectrum of pathologies that starts with simple steatosis, which can progress to non-alcoholic steatohepatitis (NASH) with the commission of other factors such as the enhancement of reactive oxygen species (ROS). Biliverdin reductase A (BVRA) reduces biliverdin to the antioxidant bilirubin, which may serve to prevent NAFLD, and possibly the progression to NASH. To further understand the role of BVRA in hepatic function, we used CRISPR-Cas9 technology to target the Blvra gene in the murine hepa1c1c7 hepatocyte cell line (BVRA KO). BVRA activity and protein levels were significantly lower in BVRA KO vs. wild-type (WT) hepatocytes. Lipid accumulation under basal and serum-starved conditions was significantly (p < 0.05) higher in BVRA KO vs. WT cells. The loss of BVRA resulted in the reduction of mitochondria number, decreased expression of markers of mitochondrial biogenesis, uncoupling, oxidation, and fusion, which paralleled reduced mitochondrial oxygen consumption. BVRA KO cells exhibited increased levels of ROS generation and decreased levels of superoxide dismutase mRNA expression. In conclusion, our data demonstrate a critical role for BVRA in protecting against lipid accumulation and oxidative stress in hepatocytes, which may serve as a future therapeutic target for NAFLD and its progression to NASH.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Caro F, Place NM, JJ Mekalanos (2019)

Analysis of lipoprotein transport depletion in Vibrio cholerae using CRISPRi.

Proceedings of the National Academy of Sciences of the United States of America, 116(34):17013-17022.

Genes necessary for the survival or reproduction of a cell are an attractive class of antibiotic targets. Studying essential genes by classical genetics, however, is inherently problematic because it is impossible to knock them out. Here, we screened a set of predicted essential genes for growth inhibition using CRISPR-interference (CRISPRi) knockdown in the human pathogen Vibrio cholerae We demonstrate that CRISPRi knockdown of 37 predicted essential genes inhibits V. cholerae viability, thus validating the products of these genes as potential drug target candidates. V. cholerae was particularly vulnerable to lethal inhibition of the system for lipoprotein transport (Lol), a central hub for directing lipoproteins from the inner to the outer membrane (OM), with many of these lipoproteins coordinating their own essential processes. Lol depletion makes cells prone to plasmolysis and elaborate membrane reorganization, during which the periplasm extrudes into a mega outer membrane vesicle or "MOMV" encased by OM which dynamically emerges specifically at plasmolysis sites. Our work identifies the Lol system as an ideal drug target, whose inhibition could deplete gram-negative bacteria of numerous proteins that reside in the periplasm.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Zhang Y, Malzahn AA, Sretenovic S, et al (2019)

The emerging and uncultivated potential of CRISPR technology in plant science.

Nature plants, 5(8):778-794.

The application of clustered regularly interspaced short palindromic repeats (CRISPR) for genetic manipulation has revolutionized life science over the past few years. CRISPR was first discovered as an adaptive immune system in bacteria and archaea, and then engineered to generate targeted DNA breaks in living cells and organisms. During the cellular DNA repair process, various DNA changes can be introduced. The diverse and expanding CRISPR toolbox allows programmable genome editing, epigenome editing and transcriptome regulation in plants. However, challenges in plant genome editing need to be fully appreciated and solutions explored. This Review intends to provide an informative summary of the latest developments and breakthroughs of CRISPR technology, with a focus on achievements and potential utility in plant biology. Ultimately, CRISPR will not only facilitate basic research, but also accelerate plant breeding and germplasm development. The application of CRISPR to improve germplasm is particularly important in the context of global climate change as well as in the face of current agricultural, environmental and ecological challenges.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Bonafont J, Mencía Á, García M, et al (2019)

Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing.

Molecular therapy : the journal of the American Society of Gene Therapy, 27(5):986-998.

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Cosentino MS, Oses C, Vázquez Echegaray C, et al (2019)

Kat6b Modulates Oct4 and Nanog Binding to Chromatin in Embryonic Stem Cells and Is Required for Efficient Neural Differentiation.

Journal of molecular biology, 431(6):1148-1159.

Chromatin remodeling is fundamental for the dynamical changes in transcriptional programs that occur during development and stem cell differentiation. The histone acetyltransferase Kat6b is relevant for neurogenesis in mouse embryos, and mutations of this gene cause intellectual disability in humans. However, the molecular mechanisms involved in Kat6b mutant phenotype and the role of this chromatin modifier in embryonic stem (ES) cells remain elusive. In this work, we show that Kat6b is expressed in ES cells and is repressed during differentiation. Moreover, we found that this gene is regulated by the pluripotency transcription factors Nanog and Oct4. To study the functional relevance of Kat6b in ES cells, we generated a Kat6b knockout ES cell line (K6b-/-) using CRISPR/Cas9. Fluorescence correlation spectroscopy analyses suggest a more compact chromatin organization in K6b-/- cells and impaired interactions of Oct4 and Nanog with chromatin. Remarkably, K6b-/- cells showed a reduced efficiency to differentiate to neural lineage. These results reveal a role of Kat6b as a modulator of chromatin plasticity, its impact on chromatin-transcription factors interactions and its influence on cell fate decisions during neural development.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Kunova Bosakova M, Nita A, Gregor T, et al (2019)

Fibroblast growth factor receptor influences primary cilium length through an interaction with intestinal cell kinase.

Proceedings of the National Academy of Sciences of the United States of America, 116(10):4316-4325.

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Wang J, Wang C, K Wang (2019)

Generation of marker-free transgenic rice using CRISPR/Cas9 system controlled by floral specific promoters.

Journal of genetics and genomics = Yi chuan xue bao, 46(1):61-64.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Wang M, Qiao J, Yu C, et al (2019)

The auxin influx carrier, OsAUX3, regulates rice root development and responses to aluminium stress.

Plant, cell & environment, 42(4):1125-1138.

In rice, there are five members of the auxin carrier AUXIN1/LIKE AUX1 family; however, the biological functions of the other four members besides OsAUX1 remain unknown. Here, by using CRISPR/Cas9, we constructed two independent OsAUX3 knock-down lines, osaux3-1 and osaux3-2, in wild-type rice, Hwayoung (WT/HY) and Dongjin (WT/DJ). osaux3-1 and osaux3-2 have shorter primary roots (PRs), decreased lateral root (LR) density, and longer root hairs (RHs) compared with their WT. OsAUX3 expression in PRs, LRs, and RHs further supports that OsAUX3 plays a critical role in the regulation of root development. OsAUX3 locates at the plasma membrane and functions as an auxin influx carrier affecting acropetal auxin transport. OsAUX3 is up-regulated in the root apex under aluminium (Al) stress, and osaux3-2 is insensitive to Al treatments. Furthermore, 1-naphthylacetic acid accented the sensitivity of WT/DJ and osaux3-2 to respond to Al stress. Auxin concentrations, Al contents, and Al-induced reactive oxygen species-mediated damage in osaux3-2 under Al stress are lower than in WT, indicating that OsAUX3 is involved in Al-induced inhibition of root growth. This study uncovers a novel pathway alleviating Al-induced oxidative damage by inhibition of acropetal auxin transport and provides a new option for engineering Al-tolerant rice species.

RevDate: 2020-03-21

Foster K, Grüschow S, Bailey S, et al (2020)

Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR-Cas immunity.

Nucleic acids research pii:5810857 [Epub ahead of print].

Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

RevDate: 2020-03-21

Binda CS, Klaver B, Berkhout B, et al (2020)

CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.

Viruses, 12(3): pii:v12030330.

Although several studies demonstrated that the HIV proviral DNA can be effectively targeted and inactivated by the CRISPR-Cas9 system, the precise inactivation mechanism has not yet been analyzed. Whereas some studies suggested efficient proviral DNA excision upon dual-gRNA/Cas9 treatment, we previously demonstrated that hypermutation of the target sites correlated with permanent virus inactivation. To better understand the mechanism underlying HIV inactivation, we analyzed the proviral DNA upon Cas9 attack with gRNA pairs. We observed that dual-gRNA targeting resulted more frequently in target site mutation than fragment excision, while fragment inversion was rarely observed. The frequencies varied for different gRNA combinations without an obvious relationship with the distance between the target sites, indicating that other gRNA and target DNA characteristics influence the DNA cleavage and repair processes.

RevDate: 2020-03-20

Hardouin P, A Goulet (2020)

Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?.

Biochemical Society transactions pii:222431 [Epub ahead of print].

Bacteriophages (phages) and their preys are engaged in an evolutionary arms race driving the co-adaptation of their attack and defense mechanisms. In this context, phages have evolved diverse anti-CRISPR proteins to evade the bacterial CRISPR-Cas immune system, and propagate. Anti-CRISPR proteins do not share much resemblance with each other and with proteins of known function, which raises intriguing questions particularly relating to their modes of action. In recent years, there have been many structure-function studies shedding light on different CRISPR-Cas inhibition strategies. As the anti-CRISPR field of research is rapidly growing, it is opportune to review the current knowledge on these proteins, with particular emphasis on the molecular strategies deployed to inactivate distinct steps of CRISPR-Cas immunity. Anti-CRISPR proteins can be orthosteric or allosteric inhibitors of CRISPR-Cas machineries, as well as enzymes that irreversibly modify CRISPR-Cas components. This repertoire of CRISPR-Cas inhibition mechanisms will likely expand in the future, providing fundamental knowledge on phage-bacteria interactions and offering great perspectives for the development of biotechnological tools to fine-tune CRISPR-Cas-based gene edition.

RevDate: 2020-03-20

Zheng Y, Li J, Wang B, et al (2020)

Endogenous Type I CRISPR-Cas: From Foreign DNA Defense to Prokaryotic Engineering.

Frontiers in bioengineering and biotechnology, 8:62.

Establishment of production platforms through prokaryotic engineering in microbial organisms would be one of the most efficient means for chemicals, protein, and biofuels production. Despite the fact that CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies have readily emerged as powerful and versatile tools for genetic manipulations, their applications are generally limited in prokaryotes, possibly owing to the large size and severe cytotoxicity of the heterogeneous Cas (CRISPR-associated) effector. Nevertheless, the rich natural occurrence of CRISPR-Cas systems in many bacteria and most archaea holds great potential for endogenous CRISPR-based prokaryotic engineering. The endogenous CRISPR-Cas systems, with type I systems that constitute the most abundant and diverse group, would be repurposed as genetic manipulation tools once they are identified and characterized as functional in their native hosts. This article reviews the major progress made in understanding the mechanisms of invading DNA immunity by type I CRISPR-Cas and summarizes the practical applications of endogenous type I CRISPR-based toolkits for prokaryotic engineering.

RevDate: 2020-03-20

Manghwar H, Li B, Ding X, et al (2020)

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off-Target Evaluation, and Strategies to Mitigate Off-Target Effects.

Advanced science (Weinheim, Baden-Wurttemberg, Germany), 7(6):1902312 pii:ADVS1528.

Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator-Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost-effectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by off-target effects. Efforts are being made to reduce the off-target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off-target effects. Here, the current GE tools, the off-target effects generated by GE technology, types of off-target effects, mechanisms of off-target effects, major concerns, and outcomes of off-target effects in plants and animals are summarized. The methods to detect off-target effects, tools for single-guide RNA (sgRNA) design, evaluation and prediction of off-target effects, and strategies to increase the on-target efficiency and mitigate the off-target impact on intended genome-editing outcomes are summarized.

RevDate: 2020-03-20

Li Q, Sapkota M, E van der Knaap (2020)

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement.

Horticulture research, 7:36 pii:258.

Directed breeding of horticultural crops is essential for increasing yield, nutritional content, and consumer-valued characteristics such as shape and color of the produce. However, limited genetic diversity restricts the amount of crop improvement that can be achieved through conventional breeding approaches. Natural genetic changes in cis-regulatory regions of genes play important roles in shaping phenotypic diversity by altering their expression. Utilization of CRISPR/Cas editing in crop species can accelerate crop improvement through the introduction of genetic variation in a targeted manner. The advent of CRISPR/Cas-mediated cis-regulatory region engineering (cis-engineering) provides a more refined method for modulating gene expression and creating phenotypic diversity to benefit crop improvement. Here, we focus on the current applications of CRISPR/Cas-mediated cis-engineering in horticultural crops. We describe strategies and limitations for its use in crop improvement, including de novo cis-regulatory element (CRE) discovery, precise genome editing, and transgene-free genome editing. In addition, we discuss the challenges and prospects regarding current technologies and achievements. CRISPR/Cas-mediated cis-engineering is a critical tool for generating horticultural crops that are better able to adapt to climate change and providing food for an increasing world population.

RevDate: 2020-03-20

Jiménez A, Hoff B, JL Revuelta (2020)

Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1.

New biotechnology pii:S1871-6784(20)30089-3 [Epub ahead of print].

CRISPR/Cas technologies constitute essential tools for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained by the existence of a specific 5'-NGG-3' PAM sequence in the target site. Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows a T-rich PAM sequence (5'-TTTN-3') to be employed and facilitates implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions with five different auxotrophic markers (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system is demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multi-CRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may affect significantly the editing efficiency of the system.

RevDate: 2020-03-20
CmpDate: 2020-03-20

Ledford H (2020)

CRISPR treatment inserted directly into the body for first time.

Nature, 579(7798):185.

RevDate: 2020-03-20
CmpDate: 2020-03-20

Huhn SC, Ou Y, Kumar A, et al (2019)

High throughput, efficacious gene editing & genome surveillance in Chinese hamster ovary cells.

PloS one, 14(12):e0218653.

Chinese hamster ovary (CHO) cells are a common tool utilized in bioproduction and directed genome engineering of CHO cells is of great interest to enhance recombinant cell lines. Until recently, this focus has been challenged by a lack of efficacious, high throughput, and low-cost gene editing modalities and screening methods. In this work, we demonstrate an improved method for gene editing in CHO cells using CRISPR RNPs and characterize the endpoints of Cas9 and ZFN mediated genetic engineering. Furthermore, we validate sequence decomposition as a cost effective, rapid, and accurate method for assessing mutants and eliminating non-clonal CHO populations using only capillary sequencing.

RevDate: 2020-03-20
CmpDate: 2020-03-20

Roy A, Zhai Y, Ortiz J, et al (2019)

Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development.

PloS one, 14(10):e0223765.

Whitefly-transmitted begomoviruses cause serious damage to many economically important food, feed, and fiber crops. Numerous vegetable crops are severely affected and chilli leaf curl virus (ChiLCV) is the most dominant and widely distributed begomovirus in chilli (Capsicum annuum) throughout the Indian subcontinent. Recently, CRISPR-Cas9 technology was used as a means to reduce geminivirus replication in infected plants. However, this approach was shown to have certain limitations such as the evolution of escape mutants. In this study, we used a novel, multiplexed guide RNA (gRNA) based CRISPR-Cas9 approach that targets the viral genome at two or more sites simultaneously. This tactic was effective in eliminating the ChiLCV genome without recurrence of functional escape mutants. Six individual gRNA spacer sequences were designed from the ChiLCV genome and in vitro assays confirmed the cleavage behaviour of these spacer sequences. Multiplexed gRNA expression clones, based on combinations of the above-mentioned spacer sequences, were developed. A total of nine-duplex and two-triplex CRISPR-Cas9 constructs were made. The efficacy of these constructs was tested for inhibition of ChiLCV infection in Nicotiana benthamiana. Results indicated that all the constructs caused a significant reduction in viral DNA accumulation. In particular, three constructs (gRNA5+4, gRNA5+2 and gRNA1+2) were most effective in reducing the viral titer and symptoms. T7E1 assay and sequencing of the targeted viral genome did not detect any escape mutants. The multiplexed genome-editing technique could be an effective way to trigger a high level of resistance against begemoviruses. To our knowledge, this is the first report of demonstrating the effectiveness of a multiplexed gRNA-based plant virus genome editing to minimize and eliminate escape mutant formation.

RevDate: 2020-03-20
CmpDate: 2020-03-20

Chen G, Abdeen AA, Wang Y, et al (2019)

A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing.

Nature nanotechnology, 14(10):974-980.

Delivery technologies for the CRISPR-Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing system often require viral vectors, which pose safety concerns for therapeutic genome editing1. Alternatively, cationic liposomal components or polymers can be used to encapsulate multiple CRISPR components into large particles (typically >100 nm diameter); however, such systems are limited by variability in the loading of the cargo. Here, we report the design of customizable synthetic nanoparticles for the delivery of Cas9 nuclease and a single-guide RNA (sgRNA) that enables the controlled stoichiometry of CRISPR components and limits the possible safety concerns in vivo. We describe the synthesis of a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating, called a nanocapsule (NC), around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. The NC is synthesized by in situ polymerization, has a hydrodynamic diameter of 25 nm and can be customized via facile surface modification. NCs efficiently generate targeted gene edits in vitro without any apparent cytotoxicity. Furthermore, NCs produce robust gene editing in vivo in murine retinal pigment epithelium (RPE) tissue and skeletal muscle after local administration. This customizable NC nanoplatform efficiently delivers CRISPR RNP complexes for in vitro and in vivo somatic gene editing.

RevDate: 2020-03-20
CmpDate: 2020-03-20

Boettcher S, Miller PG, Sharma R, et al (2019)

A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies.

Science (New York, N.Y.), 365(6453):599-604.

TP53, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common TP53 missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single-amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for TP53 missense mutations. Thus, a DNE is the primary unit of selection for TP53 missense mutations in myeloid malignancies.

RevDate: 2020-03-20
CmpDate: 2020-03-20

Wang H, Tang Z, Li T, et al (2019)

CRISPR/Cas9-Mediated Gene Knockout of ARID1A Promotes Primary Progesterone Resistance by Downregulating Progesterone Receptor B in Endometrial Cancer Cells.

Oncology research, 27(9):1051-1060.

Medroxyprogesterone (MPA) is used for the conservative treatment of endometrial cancer. Unfortunately, progesterone resistance seriously affects its therapeutic effect. The purpose of the current study was to investigate the influence of deletion of AT-rich interactive domain 1A (ARID1A) in progesterone resistance in Ishikawa cells. Ablation of ARID1A was conducted through the CRISPR/Cas9 technology. Acquired progesterone-resistant Ishikawa (Ishikawa-PR) cells were generated by chronic exposure of Ishikawa cells to MPA. The sensitivity of the parental Ishikawa, Ishikawa-PR, and ARID1A-deficient cells to MPA and/or LY294002 was determined using the Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis. In addition, Western blot analysis and reverse transcription-polymerase chain reaction was performed to evaluate the mRNA and protein expression levels of ARID1A, progesterone receptor B (PRB), and P-AKT. Both Ishikawa-PR and ARID1A knockout cells showed insensitivity to MPA, downregulation of PRB, and hyperphosphorylation of AKT compared to the parental Ishikawa cells. Pretreatment with LY294002 significantly enhanced the ability of MPA to suppress proliferation and to induce apoptosis in the parental and Ishikawa-PR cells via the inhibition of AKT activation and upregulation of PRB transcriptional activity. However, the PRB transcriptional activity and insensitivity to MPA were irreversible by LY294002 in ARID1A-deficient cells. Ablation of ARID1A is associated with low PRB expression, which serves an important role in primary progesterone resistance. Akt inhibition cannot rescue PRB or sensitize to MPA in ARID1A knockout cells. These findings suggest that ARID1A may act as a reliable biomarker to predict the response for the combination of AKT inhibitor and MPA treatment.

RevDate: 2020-03-19

Sashidhar N, Harloff HJ, Potgieter L, et al (2020)

Gene editing of three BnITPK genes in tetraploid oilseed rape leads to significant reduction of phytic acid in seeds.

Plant biotechnology journal [Epub ahead of print].

Commercialization of Brassica napus. L (oilseed rape) meal as protein diet is gaining more attention due to its well-balanced amino acid and protein contents. Phytic acid (PA) is a major source of phosphorous in plants but is considered as anti-nutritive for mono-gastric animals including humans due to its adverse effects on essential mineral absorption. The undigested PA causes eutrophication, which potentially threatens aquatic life. PA accounts to 2-5% in mature seeds of oilseed rape and is synthesized by complex pathways involving multiple enzymes. Breeding polyploids for recessive traits is challenging as gene functions are encoded by several paralogs. Gene redundancy often requires to knockout several gene copies to study their underlying effects. Therefore, we adopted CRISPR-Cas mutagenesis to knock out three functional paralogs of BnITPK. We obtained low PA mutants with an increase of free phosphorous in the canola grade spring cultivar Haydn. These mutants could mark an important milestone in rapeseed breeding with an increase in protein value and no adverse effects on oil contents.

RevDate: 2020-03-19

Kulkarni A, Yu W, Moon AS, et al (2020)

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes.

Journal of genomics, 8:30-36 pii:jgenv08p0030.

In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized in vitro using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In Anopheles gambiae, vitellogenin gene was silenced by Cas13a/Vg-crRNA, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the α- and δ-subunits of COPI genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.

RevDate: 2020-03-19

Xu S, Zhou L, Liang X, et al (2020)

Novel Cell-Virus-virophage (CVv) tripartite infection systems are discovered in a freshwater lake, Dishui Lake, in Shanghai, China.

Journal of virology pii:JVI.00149-20 [Epub ahead of print].

Virophages are small parasitic dsDNA viruses of giant dsDNA viruses infecting unicellular eukaryotes. Except for few isolated virophages characterized by parasitization mechanisms, features of virophages discovered in metagenomic data sets remain largely unknown. Here, complete genomes of seven virophages (26.6-31.5 kbp) and four large DNA viruses (190.4-392.5 kbp) are identified to co-exist in the freshwater lake, Dishui Lake, in Shanghai, China, based on environmental metagenomic investigation. Both genomic and phylogenetic analyses indicate that Dishui Lake virophages (DSLVs) are closely related to each other and other lake virophages, and Dishui Lake large DNA viruses affiliate with the micro-green algae-infecting Prasinovirus of the Phycodnaviridae (named Dishui Lake phycodnaviruses, DSLPVs) and protists (protozoa and algae)-infecting Mimiviridae (named Dishui Lake large algae virus, DSLLAV), respectively. The DSLVs possess more genes with closer homology to that of large algae viruses than to that of giant protozoa viruses. Furthermore, the DSLVs are strongly associated with large green algae viruses, including DSLPV4 and DSLLAV1, based on codon usage as well as oligonucleotide frequency and correlation analyses. Surprisingly, a non-homologous CRISPR-Cas like system is found in DSLLAV1, which appears to protect DSLLAV1 from the parasitizing of DSLV5 and 8. These results suggest novel Cell-Virus-virophage (CVv) tripartite infection systems of green algae, large green algae virus (Phycodnaviridae and Mimiviridae-related) and virophage exist in Dishui Lake, which contribute to further deep investigating of the evolutionary interaction of virophages and large algae viruses as well as of the essential roles that the CVv plays in the ecology of algae.ImportanceVirophages are small parasitizing viruses of large/giant viruses. To our knowledge, the few isolated virophages all parasitize giant protozoa viruses (Mimiviridae) for propagation and form a tripartite infection system with hosts, here named as Cell-Virus-virophage (CVv). However, the CVv system remains largely unknown in environmental metagenomic data sets. In this study, we systematically investigate the metagenomic dataset from the freshwater lake, Dishui Lake, in Shanghai, China. Consequently, four novel large algae viruses and seven virophages are discovered to co-exist in Dishui Lake. Surprisingly, a novel CVv tripartite infection system comprising green algae, large green algae virus (Phycodnaviridae and Mimiviridae-related) and virophage is identified based on genetic link, genomic signature and CRISPR system analyses. Meanwhile, a non-homologous CRISPR like system is found in Dishui Lake large algae virus, which appears to protect the virus host from the infection of DSLVs. These findings are critical to give insight into the potential significances of CVv in global evolution and ecology.

RevDate: 2020-03-19
CmpDate: 2020-03-19

Chen J, Brunner AD, Cogan JZ, et al (2020)

Pervasive functional translation of noncanonical human open reading frames.

Science (New York, N.Y.), 367(6482):1140-1146.

Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.

RevDate: 2020-03-19
CmpDate: 2020-03-19

Cuculis L, Zhao C, Abil Z, et al (2020)

Divalent cations promote TALE DNA-binding specificity.

Nucleic acids research, 48(3):1406-1422.

Recent advances in gene editing have been enabled by programmable nucleases such as transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9. However, several open questions remain regarding the molecular machinery in these systems, including fundamental search and binding behavior as well as role of off-target binding and specificity. In order to achieve efficient and specific cleavage at target sites, a high degree of target site discrimination must be demonstrated for gene editing applications. In this work, we studied the binding affinity and specificity for a series of TALE proteins under a variety of solution conditions using in vitro fluorescence methods and molecular dynamics (MD) simulations. Remarkably, we identified that TALEs demonstrate high sequence specificity only upon addition of small amounts of certain divalent cations (Mg2+, Ca2+). However, under purely monovalent salt conditions (K+, Na+), TALEs bind to specific and non-specific DNA with nearly equal affinity. Divalent cations preferentially bind to DNA over monovalent cations, which attenuates non-specific interactions between TALEs and DNA and further stabilizes specific interactions. Overall, these results uncover new mechanistic insights into the binding action of TALEs and further provide potential avenues for engineering and application of TALE- or TALEN-based systems for genome editing and regulation.

RevDate: 2020-03-19
CmpDate: 2020-03-19

Oh-Hashi K, Kohno H, Kandeel M, et al (2020)

Characterization of IRE1α in Neuro2a cells by pharmacological and CRISPR/Cas9 approaches.

Molecular and cellular biochemistry, 465(1-2):53-64.

IRE1 is the most conserved endoplasmic reticulum (ER)-resident stress sensor. Its activation not only splices XBP1 but also participates in a variety of cell signaling. We elucidated the role of IRE1α in Neuro2a cells by establishing IRE1α-deficient cells and applying four IRE1 inhibitors. IRE1α deficiency prevented almost all spliced XBP1 (sXBP1) protein expression by treatment with thapsigargin (Tg) and tunicamycin (Tm); these phenomena paralleled the values measured by our two Nanoluciferase-based IRE1 assays. However, cell viability and protein expression of other ER stress-responsive factors in the IRE1α-deficient cells were comparable to those in the parental wild-type cells with or without Tm treatment. Next, we elucidated the IRE1 inhibitory actions and cytotoxicity of four compounds: STF083010, KIRA6, 4μ8C, and toyocamycin. KIRA6 attenuated IRE1 activity in a dose-dependent manner, but it showed severe cytotoxicity even in the IRE1α-deficient cells at a low concentration. The IRE1α-deficient cells were slightly resistant to KIRA6 at 0.1 μM in both the presence and absence of ER stress; however, resistance was not observed at 0.02 μM. Treatment with only KIRA6 at 0.1 μM for 12 h remarkably induced LC3 II, an autophagic marker, in both parental and IRE1α-deficient cells. Co-treatment with KIRA6 and Tm induced LC3 II, cleaved caspase-9, and cleaved caspase-3; however, IRE1α-deficiency did not abolish the expression of these two cleaved caspases. On the other hand, KIRA6 prohibited Tm-induced ATF4 induction in an IRE1-independent manner; however, co-treatment with KIRA6 and Tm also induced LC3 II and two cleaved caspases in the ATF4-deficient Neuro2a cells. Thus, we demonstrate that IRE1α deficiency has little impact on cell viability and expression of ER stress-responsive factors in Neuro2a cells, and the pharmacological actions of KIRA6 include IRE1-independent ways.

RevDate: 2020-03-19
CmpDate: 2020-03-19

Cobb LH, Park J, Swanson EA, et al (2019)

CRISPR-Cas9 modified bacteriophage for treatment of Staphylococcus aureus induced osteomyelitis and soft tissue infection.

PloS one, 14(11):e0220421.

Osteomyelitis, or bone infection, is often induced by antibiotic resistant Staphylococcus aureus strains of bacteria. Although debridement and long-term administration of antibiotics are the gold standard for osteomyelitis treatment, the increase in prevalence of antibiotic resistant bacterial strains limits the ability of clinicians to effectively treat infection. Bacteriophages (phages), viruses that in a lytic state can effectively kill bacteria, have gained recent attention for their high specificity, abundance in nature, and minimal risk of host toxicity. Previously, we have shown that CRISPR-Cas9 genomic editing techniques could be utilized to expand temperate bacteriophage host range and enhance bactericidal activity through modification of the tail fiber protein. In a dermal infection study, these CRISPR-Cas9 phages reduced bacterial load relative to unmodified phage. Thus we hypothesized this temperate bacteriophage, equipped with the CRISPR-Cas9 bactericidal machinery, would be effective at mitigating infection from a biofilm forming S. aureus strain in vitro and in vivo. In vitro, qualitative fluorescent imaging demonstrated superiority of phage to conventional vancomycin and fosfomycin antibiotics against S. aureus biofilm. Quantitative antibiofilm effects increased over time, at least partially, for all fosfomycin, phage, and fosfomycin-phage (dual) therapeutics delivered via alginate hydrogel. We developed an in vivo rat model of osteomyelitis and soft tissue infection that was reproducible and challenging and enabled longitudinal monitoring of infection progression. Using this model, phage (with and without fosfomycin) delivered via alginate hydrogel were successful in reducing soft tissue infection but not bone infection, based on bacteriological, histological, and scanning electron microscopy analyses. Notably, the efficacy of phage at mitigating soft tissue infection was equal to that of high dose fosfomycin. Future research may utilize this model as a platform for evaluation of therapeutic type and dose, and alternate delivery vehicles for osteomyelitis mitigation.

RevDate: 2020-03-19
CmpDate: 2020-03-19

Garmendia I, Pajares MJ, Hermida-Prado F, et al (2019)

YES1 Drives Lung Cancer Growth and Progression and Predicts Sensitivity to Dasatinib.

American journal of respiratory and critical care medicine, 200(7):888-899.

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.

RevDate: 2020-03-19
CmpDate: 2020-03-19

Kim D, Luk K, Wolfe SA, et al (2019)

Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases.

Annual review of biochemistry, 88:191-220.

Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.

RevDate: 2020-03-18

Davidson AR, Lu WT, Stanley SY, et al (2020)

Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems.

Annual review of biochemistry [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems. Expected final online publication date for the Annual Review of Biochemistry, Volume 89 is June 22, 2020. Please see for revised estimates.

RevDate: 2020-03-18

Banas K, Rivera-Torres N, Bialk P, et al (2020)

Kinetics of nuclear uptake and site-specific DNA cleavage during CRISPR-directed gene editing in solid tumor cells.

Molecular cancer research : MCR pii:1541-7786.MCR-19-1208 [Epub ahead of print].

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-directed gene editing is approaching clinical implementation in cancer. Thus, it is imperative to define the molecular framework upon which safe and efficacious therapeutic strategies can be built. Two important reaction parameters include the biological time frame within which the CRISPR/Cas complex enters the nucleus and executes gene editing, and the method of discrimination that the CRISPR/Cas complex utilizes to target tumor cell, but not normal cell, genomes. We are developing CRISPR-directed gene editing for the treatment of non-small cell lung carcinoma (NSCLC) focusing on disabling Nuclear Factor Erythroid 2-Related Factor-Like (NRF2), a transcription factor that regulates chemoresistance and whose genetic disruption would enhance chemosensitivity. In this report, we define the time frame of cellular events that surround the initialization of CRISPR-directed gene editing as a function of the nuclear penetration and the execution of NRF2 gene disruption. We also identify a unique protospacer adjacent motif (PAM) that facilitates site-specific cleavage of the NRF2 gene present only in tumor genomes. Implications: Our results begin to set a scientifically meritorious foundation for the exploitation of CRISPR-directed gene editing as an augmentative therapy for lung cancer and other solid tumors.

RevDate: 2020-03-18

Xie JY, Wei JX, Lv LH, et al (2020)

Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma.

Cell communication and signaling : CCS, 18(1):46 pii:10.1186/s12964-020-00535-8.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. Exosomes are small extracellular vesicles secreted by most cell types and contribute to cell-to-cell communication by delivering functional cargo to recipient cells. The expression of ANGPT2 in tumor-derived exosomes remains unknown.

METHODS: We detected the ANGPT2 expression in HCC-derived exosomes by immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by confocal laser scanning microscopy, co-immunoprecipitation and immunoblotting. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay.

RESULTS: We found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC.

CONCLUSIONS: Taken together, these results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy. Video Abstract.


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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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

Electronic Scholarly Publishing
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Bellingham, WA 98226

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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).


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


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.

ESP Picks from Around the Web (updated 07 JUL 2018 )