CRISPR nuclease off-target activity and mitigation strategies.

IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Frontiers in genome editing Pub Date : 2022-11-10 eCollection Date: 2022-01-01 DOI:10.3389/fgeed.2022.1050507
Beeke Wienert, M Kyle Cromer
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引用次数: 0

Abstract

The discovery of CRISPR has allowed site-specific genomic modification to become a reality and this technology is now being applied in a number of human clinical trials. While this technology has demonstrated impressive efficacy in the clinic to date, there remains the potential for unintended on- and off-target effects of CRISPR nuclease activity. A variety of in silico-based prediction tools and empirically derived experimental methods have been developed to identify the most common unintended effect-small insertions and deletions at genomic sites with homology to the guide RNA. However, large-scale aberrations have recently been reported such as translocations, inversions, deletions, and even chromothripsis. These are more difficult to detect using current workflows indicating a major unmet need in the field. In this review we summarize potential sequencing-based solutions that may be able to detect these large-scale effects even at low frequencies of occurrence. In addition, many of the current clinical trials using CRISPR involve ex vivo isolation of a patient's own stem cells, modification, and re-transplantation. However, there is growing interest in direct, in vivo delivery of genome editing tools. While this strategy has the potential to address disease in cell types that are not amenable to ex vivo manipulation, in vivo editing has only one desired outcome-on-target editing in the cell type of interest. CRISPR activity in unintended cell types (both on- and off-target) is therefore a major safety as well as ethical concern in tissues that could enable germline transmission. In this review, we have summarized the strengths and weaknesses of current editing and delivery tools and potential improvements to off-target and off-tissue CRISPR activity detection. We have also outlined potential mitigation strategies that will ensure that the safety of CRISPR keeps pace with efficacy, a necessary requirement if this technology is to realize its full translational potential.

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CRISPR 核酸酶脱靶活性及缓解策略。
CRISPR 的发现使特定位点的基因组改造成为现实,这项技术目前已应用于多项人体临床试验。迄今为止,这项技术在临床上已显示出令人印象深刻的疗效,但 CRISPR 核酸酶活性仍有可能产生非预期的靶上和靶下效应。目前已开发出多种基于硅学的预测工具和根据经验得出的实验方法,用于识别最常见的意外效应--与引导 RNA 同源的基因组位点上的小规模插入和缺失。不过,最近也有大规模畸变的报道,如易位、倒位、缺失,甚至染色体反常。使用当前的工作流程很难检测到这些畸变,这表明该领域的主要需求尚未得到满足。在这篇综述中,我们总结了潜在的基于测序的解决方案,这些方案即使发生频率较低,也能检测到这些大规模效应。此外,目前许多使用CRISPR的临床试验都涉及患者自身干细胞的体外分离、改造和再移植。然而,人们对在体内直接提供基因组编辑工具的兴趣日益浓厚。虽然这种策略有可能解决无法进行体内外操作的细胞类型的疾病问题,但体内编辑只有一种预期结果--在感兴趣的细胞类型中进行靶向编辑。因此,CRISPR 在非预期细胞类型中的活性(包括靶上和非靶上)是一个主要的安全性问题,也是可能导致种系传播的组织中的伦理问题。在这篇综述中,我们总结了当前编辑和传递工具的优缺点,以及对靶外和组织外 CRISPR 活性检测的潜在改进。我们还概述了潜在的缓解策略,这些策略将确保 CRISPR 的安全性与有效性保持同步,这是该技术要充分发挥其转化潜力的必要条件。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
7.00
自引率
0.00%
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审稿时长
13 weeks
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