Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells
M. Kyle CromerValentin BarsanErich JaegerMengchi WangJessica P. HamptonFeng ChenDrew KennedyJenny XiaoIrina KhrebtukovaAna GranatTiffany TruongMatthew H. Porteus
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Abstract As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here, we employ a clinical next generation sequencing (NGS) workflow to achieve high sequencing depth and detect ultra-low frequency variants across exons of genes associated with cancer, all exons, and genome wide. In three separate primary human hematopoietic stem and progenitor cell (HSPC) donors assessed in technical triplicates, we electroporated high-fidelity Cas9 protein targeted to three loci (AAVS1, HBB , and ZFPM2 ) and harvested genomic DNA at days 4 and 10. Our results demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP in a gRNA spacer sequence is sufficient to eliminate Cas9 off-target activity in primary, repair-competent human HSPCs.Zinc finger nuclease
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Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo genome engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed.
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Genome editing comprises the use of customizable engineered nucleases for the genetic modification of cells. Two commonly used systems are transcription activator-like effector nucleases (TALEN) and the CRISPR/Cas system. During this study, genome editing tools were applied for the modification of three genes (JAK3, RAG1, RAG2) playing crucial roles in the immune system, with the aim of generating immunodeficient pigs.
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This review will highlight some of the recent advances in genome engineering with applications for both clinical and basic science investigations of HIV-1.Over the last year, the field of HIV cure research has seen major breakthroughs with the success of the first phase I clinical trial involving gene editing of CCR5 in patient-derived CD4(+) T cells. This first human use of gene-editing technology was accomplished using zinc finger nucleases (ZFNs). Zinc finger nucleases and the advent of additional tools for genome engineering, including transcription activator-like effector nucleases (TALENS) and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, have made gene editing remarkably simple and affordable. Here we will discuss the different gene-editing technologies, the use of gene editing in HIV research over the past year, and potential applications of gene editing for both in-vitro and in-vivo studies.Genome-engineering technologies have rapidly progressed over the past few years such that these systems can be easily applied in any laboratory for a variety of purposes. For HIV-1, upcoming clinical trials will determine if gene editing can provide the long-awaited functional cure. In addition, manipulation of host genomes, whether in vivo or in vitro, can facilitate development of better animal models and culture methods for studying HIV-1 transmission, pathogenesis, and virus-host interactions.
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The most significant challenges to improving yield in agriculture science need progressive improvement with novel strategies. The modern gene editing scenario must focus on reducing the infectious plant diseases caused by plant pathogens. Targeted plant genome engineering enables optimal modifications in commercial plant varieties by incorporating effective alternative gene editing methods over other traditional approaches. Genome editing technology is a multipurpose approach that rapidly progressed in plant biology to alter the phenotype and genotype of the species. Despite its advantages, traditional genome modification could not meet the recent demands for site-directed mutagenesis, high effectiveness, and retrofitting of artificial endonucleases 3 . Current gene editing technologies have been subdivided into three kinds of cleavage systems, which are Transcription activator-like effector nucleases TALEN, Zinc Finger nucleases ZFN, CRISPR Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated protein). These methods lead to single-stranded or double-stranded mutation through homologous recombination. Hence, this Review is motivated by the advantages of the gene editing tools in developing the plant immune system.
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Abstract CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR–Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens , a comprehensive genome-wide investigation of off-target mutations using either a CRISPR–Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.
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Targeted genomic editing technologies use programmable DNA nucleases to cleave genomic target sites, thus inducing targeted mutations in the genomes. The newly prevailed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system that consists of the Cas9 nuclease and single guide RNA (sgRNA) has the advantages of simplicity and high efficiency as compared to other programmable DNA nuclease systems such as zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). Currently, a number of cases have been reported on the application of the CRISPR/Cas9 genomic editing technology in plants. In this review, we summarize the strategies for preparing the Cas9 and sgRNA expression constructs, the transformation method for obtaining targeted mutations, the efficiency and features of the resulting mutations and the methods for detecting or genotyping of the mutation sites. We also discuss the existing problems and perspectives of CRISPR/Cas9-based genomic editing in plants.
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