Predicting the mutations generated by repair of Cas9-induced double-strand breaks
Predicting the mutations generated by repair of Cas9-induced double-strand breaks
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DOI：
10.1038/nbt.4317
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发表时间：
2019-01-01
影响因子：
46.9
通讯作者：
Parts, Leopold
中科院分区：
文献类型：
--
作者：
Allen, Felicity;Crepaldi, Luca;Parts, Leopold
细胞修复crispr - cas9产生的双链断裂所产生的DNA突变决定了其表型效应。众所周知，突变结果不是随机的，而是取决于目标位置的DNA序列。在这里，我们系统地研究了侧翼DNA序列对修复结果的影响，通过测量合成构建体中40000个引导rna （gRNAs）产生的编辑。我们在一系列遗传背景下进行实验，并使用替代的CR ISPR-Cas9试剂。我们总共收集了bb1010(9)个突变结果的数据。大多数可复制的突变是单个碱基的插入，短缺失或更长的微同源介导的缺失。每个gRNA对特定的结果有单独的细胞系依赖的偏倚。我们发现了产生突变的序列决定因素，并利用这些来得出Cas9编辑结果的预测因子。提高对序列修复的理解将有助于更好地设计基因编辑实验。
The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CR ISPR-Cas9 reagents. In total, we gathered data for >10(9) mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.