Evaluation of Homology-Independent CRISPR-Cas9 Off-Target Assessment Methods.
Evaluation of Homology-Independent CRISPR-Cas9 Off-Target Assessment Methods.
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DOI：
10.1089/crispr.2020.0053
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发表时间：
2020-12
期刊：
影响因子：
--
通讯作者：
Kernytsky A
中科院分区：
文献类型：
--
作者：
Chaudhari HG;Penterman J;Whitton HJ;Spencer SJ;Flanagan N;Lei Zhang MC;Huang E;Khedkar AS;Toomey JM;Shearer CA;Needham AW;Ho TW;Kulman JD;Cradick TJ;Kernytsky A
通过CRISPR-Cas基因编辑改变基因组的能力已经彻底改变了生物研究、生物技术和医学。然而，该技术的广泛治疗应用将需要通过基于同源性的预测以及检测脱靶编辑的可靠方法对脱靶编辑进行彻底的临床前评估。存在几种脱靶位点提名分析，但需要仔细比较以确定其相对优势和劣势。在本研究中，用化脓性链球菌Cas9和8种不同预测混杂水平的引导rna处理HEK293T细胞，以比较三种不依赖同源的脱靶命名方法的性能：基于细胞的检测、guide -seq以及生化检测CIRCLE-seq和SITE-seq。这三种方法通过对75,000个同源指定位点进行杂交捕获和高通量测序来进行基准测试，提供了迄今为止这些方法最全面的评估。三种方法在提名序列确认的脱靶位点方面表现相似，但在提名的位点总数上存在较大差异。当与同源依赖的提名方法和测序确认相结合时，所有三种脱靶提名方法都提供了脱靶活性的综合评估。GUIDE-seq的低假阳性率及其信号与观察到的编辑高度相关，突出了它在体外CRISPR-Cas治疗中提名脱靶位点的适用性。
The ability to alter genomes specifically by CRISPR-Cas gene editing has revolutionized biological research, biotechnology, and medicine. Broad therapeutic application of this technology, however, will require thorough preclinical assessment of off-target editing by homology-based prediction coupled with reliable methods for detecting off-target editing. Several off-target site nomination assays exist, but careful comparison is needed to ascertain their relative strengths and weaknesses. In this study, HEK293T cells were treated with Streptococcus pyogenes Cas9 and eight guide RNAs with varying levels of predicted promiscuity in order to compare the performance of three homology-independent off-target nomination methods: the cell-based assay, GUIDE-seq, and the biochemical assays CIRCLE-seq and SITE-seq. The three methods were benchmarked by sequencing 75,000 homology-nominated sites using hybrid capture followed by high-throughput sequencing, providing the most comprehensive assessment of such methods to date. The three methods performed similarly in nominating sequence-confirmed off-target sites, but with large differences in the total number of sites nominated. When combined with homology-dependent nomination methods and confirmation by sequencing, all three off-target nomination methods provide a comprehensive assessment of off-target activity. GUIDE-seq's low false-positive rate and the high correlation of its signal with observed editing highlight its suitability for nominating off-target sites for ex vivo CRISPR-Cas therapies.
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影响因子：
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DOI：
10.1073/pnas.1007983107
发表时间：
2010-07-13
影响因子：
11.1
作者：
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作者：
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