Добірка наукової літератури з теми "CRISPR/Cas, genome editing, genetic engineering, off-target assessment"

The industrially important non-conventional yeast Komagataella phaffii suffers from low rates of homologous recombination, making site specific genetic engineering tedious. Therefore, genome editing using CRISPR/Cas represents a simple and efficient alternative. To characterize on- and off-target mutations caused by CRISPR/Cas9 followed by non-homologous end joining repair, we chose a diverse set of CRISPR/Cas targets and conducted whole genome sequencing on 146 CRISPR/Cas9 engineered single colonies. We compared the outcomes of single target CRISPR transformations to double target experiments. Furthermore, we examined the extent of possible large deletions by targeting a large genomic region, which is likely to be non-essential. The analysis of on-target mutations showed an unexpectedly high number of large deletions and chromosomal rearrangements at the CRISPR target loci. We also observed an increase of on-target structural variants in double target experiments as compared to single target experiments. Targeting of two loci within a putatively non-essential region led to a truncation of chromosome 3 at the target locus in multiple cases, causing the deletion of 20 genes and several ribosomal DNA repeats. The identified de novo off-target mutations were rare and randomly distributed, with no apparent connection to unspecific CRISPR/Cas9 off-target binding sites.

CRISPR/Cas system, a microbial adaptive immune system, has rapidly transformed the ways researchers can interrogate the genome. CRISPR has many advantages over traditional methods such as Transcription activator-like effector nucleases (TALEN) and Zinc-finger nucleases (ZFN). Since CRISPR discovery as an adaptive immune system used by bacterial against viruses, it has been repurposed to help in many different genome-related studies such as gene knocking in and out, gene expression upregulation and downregulation. Also CRISPR holds vast therapeutic potential for the management of genetic disorders by straight modifying disease-causing mutations. Although the Cas9 protein has been revealed to attach and cleave DNA at off-target sites, the field of Cas9 specificity is quickly progressing, with marked modifying in guide RNA choice, protein and guide engineering, innovative enzymes, and off-target recognition methods. In current review we mostly focus on CRISPR unique ability in gene activation/ upregulation, which has wide applications in different aspects such as gene studies, stem cell differentiation, and trans-differentiation. Compared to other gene activation methods such as viral gene overexpression, TALEN and ZFN, CRISPR offers many benefits such as easy designing and high precision.

Recent advances in programmable nucleases including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) have propelled genome editing from explorative research to clinical and industrial settings. Each technology, however, features distinct modes of action that unevenly impact their applicability across the entire genome and are often tested under significantly different conditions. While CRISPR-Cas is currently leading the field due to its versatility, quick adoption, and high degree of support, it is not without limitations. Currently, no technology can be regarded as ideal or even applicable to every case as the context dictates the best approach for genetic modification within a target organism. In this review, we implement a four-pillar framework (context, feasibility, efficiency, and safety) to assess the main genome editing platforms, as a basis for rational decision-making by an expanding base of users, regulators, and consumers. Beyond carefully considering their specific use case with the assessment framework proposed here, we urge stakeholders interested in genome editing to independently validate the parameters of their chosen platform prior to commitment. Furthermore, safety across all applications, particularly in clinical settings, is a paramount consideration and comprehensive off-target detection strategies should be incorporated within workflows to address this. Often neglected aspects such as immunogenicity and the inadvertent selection of mutants deficient for DNA repair pathways must also be considered.

The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering. A promising method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA, tracrRNA and sgRNA) containing modified nucleotides (2’-O-methyl, 2’-fluoro, LNA—locked nucleic acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide RNAs. The replacement of ribonucleotides with 2’-fluoro modified or LNA nucleotides increased the lifetime of the crRNAs, while other types of modification did not change their nuclease resistance. Modification of crRNA or tracrRNA preserved the efficacy of the CRISPR/Cas9 system. Otherwise, the CRISPR/Cas9 systems with modified sgRNA showed a remarkable loss of DNA cleavage efficacy. The kinetic constant of DNA cleavage was higher for the system with 2’-fluoro modified crRNA. The 2’-modification of crRNA also decreased the off-target effect upon in vitro dsDNA cleavage.

ABSTRACT Motivation Clustered regularly interspaced short palindromic repeats (CRISPR) technologies allow for facile genomic modification in a site-specific manner. A key step in this process is the in silico design of single guide RNAs to efficiently and specifically target a site of interest. To this end, it is necessary to enumerate all potential off-target sites within a given genome that could be inadvertently altered by nuclease-mediated cleavage. Currently available software for this task is limited by computational efficiency, variant support or annotation, and assessment of the functional impact of potential off-target effects. Results To overcome these limitations, we have developed CRISPRitz, a suite of software tools to support the design and analysis of CRISPR/CRISPR-associated (Cas) experiments. Using efficient data structures combined with parallel computation, we offer a rapid, reliable, and exhaustive search mechanism to enumerate a comprehensive list of putative off-target sites. As proof-of-principle, we performed a head-to-head comparison with other available tools on several datasets. This analysis highlighted the unique features and superior computational performance of CRISPRitz including support for genomic searching with DNA/RNA bulges and mismatches of arbitrary size as specified by the user as well as consideration of genetic variants (variant-aware). In addition, graphical reports are offered for coding and non-coding regions that annotate the potential impact of putative off-target sites that lie within regions of functional genomic annotation (e.g. insulator and chromatin accessible sites from the ENCyclopedia Of DNA Elements [ENCODE] project). Availability and implementation The software is freely available at: https://github.com/pinellolab/CRISPRitzhttps://github.com/InfOmics/CRISPRitz. Supplementary information Supplementary data are available at Bioinformatics online.

AbstractThe discovery of an adaptive immune system especially in archae and bacteria, CRISPR/Cas has revolutionized the field of agriculture and served as a potential gene editing tool, producing great excitement to the molecular scientists for the improved genetic manipulations. CRISPR/Cas9 is a RNA guided endonuclease which is popular among its predecessors ZFN and TALEN’s. The utilities of CRISPR from its predecessors is the use of short RNA fragments to locate target and breaking the double strands which avoids the need of protein engineering, thus allowing time efficiency measure for gene editing. It is a simple, flexible and highly efficient programmable DNA cleavage system that can be modified for widespread applications like knocking out the genes, controlling transcription, modifying epigenomes, controlling genome-wide screens, modifying genes for disease and stress tolerance and imaging chromosomes. However, gene cargo delivery system, off target cutting and issues on the safety of living organisms imposes major challenge to this system. Several attempts have been done to rectify these challenges; using sgRNA design software, cas9 nickases and other mutants. Thus, further addressing these challenges may open the avenue for CRISPR/cas9 for addressing the agriculture related problems.

Chimeric antigen receptor T (CART) cells are engineered with an artificial receptor which redirects T cells to recognize cancer cells expressing a particular surface antigen. CART cell therapy has been astonishingly successful at eradicating certain B cell malignancies, but relapse is common, and efficacy is lacking in many cancers. Gene editing of CART cells is being investigated to enhance efficacy and safety and to develop off-the-shelf products. Currently, genome engineering tools used to modify CART cells include zinc finger nucleases, transposons, TALENs, and CRISPR-Cas9. CRISPR-Cas9 uses a trans-activating (tracrRNA): CRISPR RNA (crRNA) duplex to trigger imprecise DNA repair through targeted double stranded breaks, causing indels and often resulting in loss of protein function. Gene-edited CART cells have entered the clinic to provide an allogeneic cell source (TALEN TCRα knockout), safer treatment (CRISPR-Cas9 GM-CSF knockout), and resistance to exhaustion (CRISPR-Cas9 PD-1 knockout). CRISPR-Cas9 PD-1 knockout (PD-1k/o) CART cells were well-tolerated in a first-in-human clinical trial. However, clinically tested CRISPR-Cas9-edited CART cells showed only modest loss of function (~25%) of PD-1 upon infusion. Additionally, off-target editing has been observed in the clinic and remains a concern. We hypothesized that using next-generation CRISPR-Cas12a systems will result in enhanced editing efficiency and precision. CRISPR-Cas12a has a smaller protein component than CRISPR-Cas9, uses a single crRNA without a tracrRNA for simplified delivery and leaves staggered 5' overhangs. These properties, along with lower intrinsic off-target activity than Cas9, render Cas12a a powerful gene editing tool. First, we compared the knockout efficiency of Cas9 and Cas12a in three therapeutically relevant genetic targets in T cells by delivering ribonucleoprotein complexes containing the crRNA and Cas protein of interest. We showed that Cas12a more effectively knocked out CD3, GM-CSF, and PD-1 expression compared to Cas9 (Figure 1A), demonstrating the potential of Cas12a in further genetically editing T cell therapies. We then used electroporation with Cas9 and Cas12a to generate PD-1k/o in lentivirally transduced CD19-targeted CART (CART19) cells with the aim of making exhaustion-resistant CART19 cells through CRISPR gene editing. CART19 and PD-1k/o CART19 cells were repeatedly stimulated with CD19+ NALM6 target cells for one week, and exhaustion marker expression was measured over time with flow cytometry. The expression of CTLA4, TIM3, and LAG3 were similar between CART19 groups, but PD-1 expression was lower in Cas9 PD-1k/o CART19 cells and almost completely eradicated in Cas12a PD-1k/o CART19 cells compared to wildtype or mock shocked CART19 cells (Figure 1B). We then compared the functionality of wildtype, mock shocked, and Cas9 or Cas12a PD-1k/o CART19 cells in vitro to ensure that neither the electroporation process nor PD-1 knockout impaired CART19 cell antitumor activity. Over a range of effector-to-target ratios and with repeated stimulation with target cells, cytotoxicity was comparable across all CART19 cell groups (Figure 1C). All CART19 cell groups demonstrated robust proliferation in response to both nonspecific and antigen-specific stimulation and over one week of repeated antigen stimulation with NALM6 target cells (Figure 1D). We also confirmed that all CART19 cell groups demonstrated strong degranulation and cytokine production in response to nonspecific and antigen-specific stimulation, regardless of electroporation or PD-1 knockout (Figure 1E). In summary, our data demonstrate that Cas12a can be used as a gene editing tool to efficiently knock out therapeutically relevant genes in CART19 cell therapy. Additionally, Cas12a demonstrated improved knockout efficiency over Cas9 in three different genomic targets. PD-1 knockout via Cas9 or Cas12a reduced PD-1 expression on the CART19 cell surface, and PD-1 expression was almost completely ablated with Cas12a gene editing. Electroporation and PD-1 knockout did not impact the effector functions of the CART19 cells, including cytotoxicity, degranulation, cytokine secretion, or proliferation. In vivo studies assessing the antitumor efficacy and CART19 cell persistence are ongoing. Overall, Cas12a is a promising, efficient method of gene knockout to enhance the safety and efficacy of CART cells. Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Kenderian:MorphoSys: Research Funding; Sunesis: Research Funding; Tolero: Research Funding; BMS: Research Funding; Juno: Research Funding; Gilead: Research Funding; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Lentigen: Research Funding.

AbstractAquaculture is becoming the primary source of seafood for human diets, and farmed fish aquaculture is one of its fastest growing sectors. The industry currently faces several challenges including infectious and parasitic diseases, reduced viability, fertility reduction, slow growth, escapee fish and environmental pollution. The commercialization of the growth-enhanced AquAdvantage salmon and the CRISPR/Cas9-developed tilapia (Oreochromis niloticus) proffers genetic engineering and genome editing tools, e.g. CRISPR/Cas, as potential solutions to these challenges. Future traits being developed in different fish species include disease resistance, sterility, and enhanced growth. Despite these notable advances, off-target effect and non-clarification of trait-related genes among other technical challenges hinder full realization of CRISPR/Cas potentials in fish breeding. In addition, current regulatory and risk assessment frameworks are not fit-for purpose regarding the challenges of CRISPR/Cas notwithstanding that public and regulatory acceptance are key to commercialization of products of the new technology. In this study, we discuss how CRISPR/Cas can be used to overcome some of these limitations focusing on diseases and environmental release in farmed fish aquaculture. We further present technical limitations, regulatory and risk assessment challenges of the use of CRISPR/Cas, and proffer research strategies that will provide much-needed data for regulatory decisions, risk assessments, increased public awareness and sustainable applications of CRISPR/Cas in fish aquaculture with emphasis on Atlantic salmon (Salmo salar) breeding.

Over the past decades, progression in genetic element manipulation, and consequently, the treatment of diseases has been remarkable. It is worth noting that these genetic manipulations perform at different levels, including DNA and RNA. The earlier genomic editing techniques, including MN, ZFN , TALEN , performing their functions by creating double-stranded breaks (DSBs), and after breakage, the cell tries to repair the breakage through two systems, homologous recombination and non-homologous end joining. CRISPR/Cas technology has been discovered recently, and has become the most widely used genome-editing tool, mainly due to its capabilities and those added to this through the genetic engineering. In this study, we aimed to introduce a variety of CRISPR classes in the elementary parts, and then the modified CRISPR systems developed to increase the efficiency and specificity of the system and provide acceptable results will be introduced. In this study, for three months in the fall and winter, Pubmed and Web of science sites searched for keywords such as CRISPR, Types of CRISPR, gRNA, Cas9, and CRISPR-Cas9 nickase that eventually resulted in about four hundred Sixty-one articles, and some of these articles after closer study, reviewed in this article. Genetic engineering techniques have successfully transformed this system into the most efficient genome editing tool in recent years. Researchers are working on a system to treat various diseases by resolving problems such as high specificity, cutting off non-target sites, how to move to a cell, and setting up a proper repair system.

Cancer is one of the most leading causes of mortalities worldwide. It is caused by the accumulation of genetic and epigenetic alterations in 2 types of genes: tumor suppressor genes (TSGs) and proto-oncogenes. In recent years, development of the clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized genome engineering for different cancer research ranging for research ranging from fundamental science to translational medicine and precise cancer treatment. The CRISPR/CRISPR associated proteins (CRISPR/Cas) are prokaryote-derived genome editing systems that have enabled researchers to detect, image, manipulate and annotate specific DNA and RNA sequences in various types of living cells. The CRISPR/Cas systems have significant contributions to discovery of proto-oncogenes and TSGs, tumor cell epigenome normalization, targeted delivery, identification of drug resistance mechanisms, development of high-throughput genetic screening, tumor models establishment, and cancer immunotherapy and gene therapy in clinics. Robust technical improvements in CRISPR/Cas systems have shown a considerable degree of efficacy, specificity, and flexibility to target the specific locus in the genome for the desired applications. Recent developments in CRISPRs technology offers a significant hope of medical cure against cancer and other deadly diseases. Despite significant improvements in this field, several technical challenges need to be addressed, such as off-target activity, insufficient indel or low homology-directed repair (HDR) efficiency, in vivo delivery of the Cas system components, and immune responses. This study aims to overview the recent technological advancements, preclinical and perspectives on clinical applications of CRISPR along with their advantages and limitations. Moreover, the potential applications of CRISPR/Cas in precise cancer tumor research, genetic, and other precise cancer treatments discussed.