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.

中文翻译：

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同源无关的 CRISPR-Cas9 脱靶评估方法的评估


通过 CRISPR-Cas 基因编辑专门改变基因组的能力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来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测定，但需要仔细比较以确定它们的相对优点和缺点。在这项研究中，HEK293T 细胞用化脓性链球菌Cas9 和 8 个具有不同预测混杂水平的指导 RNA 进行处理，以比较三种同源独立的脱靶提名方法的性能：基于细胞的测定、GUIDE-seq 和生化分析 CIRCLE-seq 和 SITE-seq。通过使用混合捕获和高通量测序对 75,000 个同源提名位点进行测序，对这三种方法进行了基准测试，从而提供了迄今为止对此类方法最全面的评估。这三种方法在提名序列确认的脱靶位点方面表现相似，但提名位点总数存在很大差异。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可以对脱靶活性进行全面评估。 GUIDE-seq 的低假阳性率及其信号与观察到的编辑的高度相关性凸显了它适合为离体CRISPR-Cas 疗法指定脱靶位点。