Discovery Sciences, R&D, AstraZeneca, Cambridge, UK.
Wellcome Sanger Institute, Hinxton, Cambridge, CB10 1RQ, UK.
Nat Commun. 2020 Aug 17;11(1):4132. doi: 10.1038/s41467-020-17952-5.
Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.
使用 CRISPR-Cas9 进行精确的基因组编辑是治疗遗传疾病的一种很有前途的方法，尽管脱靶编辑仍然是一个重大的安全问题。长度短于 16 个核苷酸的向导 RNA 可以有效地将 Cas9 招募到基因组中的互补位点，但不允许 Cas9 核酸酶活性。在这里，我们描述了 CRISPR 向导 RNA 辅助减少损伤（CRISPR GUARD），这是一种通过与靶向向导 RNA 竞争，共同递送靶向非靶位点的短向导 RNA 来保护非靶位点的方法。CRISPR GUARD 减少了脱靶诱变，同时保留了 Cas9 和碱基编辑器的靶向编辑效率。然而，我们发现，如果短向导 RNA 内的脱氨酶活性窗口包含胞嘧啶，它们也可以支持碱基编辑。我们通过体外研究和高通量筛选探索了这种方法的设计规则和普遍性，揭示了 CRISPR GUARD 是一种可快速实施的策略，可提高大多数基因组位点的基因组编辑特异性。最后，我们创建了一个用于 CRISPR GUARD 设计的在线工具。
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