CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs
CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs
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DOI：
10.1038/s41467-020-17952-5
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发表时间：
2020-08-17
影响因子：
16.6
通讯作者：
Taylor, Benjamin J. M.
中科院分区：
文献类型：
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作者：
Coelho, Matthew A.;De Braekeleer, Etienne;Taylor, Benjamin J. M.
使用CRISPR-Cas9进行精确的基因组编辑是一种有希望的遗传疾病治疗途径，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有效地将Cas9募集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辅助的损伤减少（CRISPR GUARD）作为一种用于通过与在靶向导RNA竞争共递送针对脱靶基因座的短向导RNA来保护脱靶位点的方法。CRISPR GUARD减少脱靶诱变，同时保留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在脱氨酶活性窗口内含有胞嘧啶，则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通用性，揭示了CRISPR GUARD是一种可快速实施的策略，可以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CRISPR GUARD设计的在线工具。脱靶编辑仍然是CRISPR-Cas9治疗应用的一个问题。在这里，作者介绍了CRISPR GUARD，它使用非常短的非切割gRNA来防止在脱靶位点进行编辑。
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. Off-target editing remains a concern for therapeutic applications of CRISPR-Cas9. Here the authors present CRISPR GUARD, which uses very short non-cleaving gRNAs to prevent editing at off-target sites.