Off-Target Analysis in Gene Editing and Applications for Clinical Translation of CRISPR/Cas9 in HIV-1 Therapy.
Off-Target Analysis in Gene Editing and Applications for Clinical Translation of CRISPR/Cas9 in HIV-1 Therapy.
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DOI：
10.3389/fgeed.2021.673022
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发表时间：
2021
影响因子：
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通讯作者：
Wigdahl B
中科院分区：
文献类型：
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作者：
Atkins A;Chung CH;Allen AG;Dampier W;Gurrola TE;Sariyer IK;Nonnemacher MR;Wigdahl B
随着基因组编辑核酸酶走向更广泛的临床应用，定义其特异性和效率的限制的需求增加。已经开发了多种用于核酸酶切割检测的方法，允许靶向景观的全基因组调查和核酸酶诱导的双链断裂的多种修复结果的检测。每种方法都具有与靶位点捕获手段、靶富集机制、细胞环境、错误发现和细胞中真正脱靶切割位点的验证有关的优点和缺点。本文综述了不同类型的脱靶切割检测系统的优势、局限性和起源，包括锚定引物富集（GUIDE-seq）、原位检测（布利斯）、体外选择文库（CIRCLE-seq）、染色质免疫沉淀（ChIP）（DISCOVER-Seq）、易位测序（LAM PCR HTGTS）和体外基因组DNA消化（Digenome-seq和SITE-Seq）。重点放在具体的修改，使当代技术的性能比他们的前辈和技术的比较性能不同的应用程序。这些技术的临床相关性在评估新型CRISPR/Cas9 HIV-1治疗策略的安全性的背景下进行了讨论。随着最近使用CRISPR/Cas9分别在人源化小鼠和非人灵长类动物中成功抑制HIV-1和SIV-1病毒，严格探索潜在的脱靶效应至关重要。这种分析将受益于本审查中讨论的技术的应用。
As genome-editing nucleases move toward broader clinical applications, the need to define the limits of their specificity and efficiency increases. A variety of approaches for nuclease cleavage detection have been developed, allowing a full-genome survey of the targeting landscape and the detection of a variety of repair outcomes for nuclease-induced double-strand breaks. Each approach has advantages and disadvantages relating to the means of target-site capture, target enrichment mechanism, cellular environment, false discovery, and validation of bona fide off-target cleavage sites in cells. This review examines the strengths, limitations, and origins of the different classes of off-target cleavage detection systems including anchored primer enrichment (GUIDE-seq), in situ detection (BLISS), in vitro selection libraries (CIRCLE-seq), chromatin immunoprecipitation (ChIP) (DISCOVER-Seq), translocation sequencing (LAM PCR HTGTS), and in vitro genomic DNA digestion (Digenome-seq and SITE-Seq). Emphasis is placed on the specific modifications that give rise to the enhanced performance of contemporary techniques over their predecessors and the comparative performance of techniques for different applications. The clinical relevance of these techniques is discussed in the context of assessing the safety of novel CRISPR/Cas9 HIV-1 curative strategies. With the recent success of HIV-1 and SIV-1 viral suppression in humanized mice and non-human primates, respectively, using CRISPR/Cas9, rigorous exploration of potential off-target effects is of critical importance. Such analyses would benefit from the application of the techniques discussed in this review.