Allen Felicity, Crepaldi Luca, Alsinet Clara, Strong Alexander J, Kleshchevnikov Vitalii, De Angeli Pietro, Páleníková Petra, Khodak Anton, Kiselev Vladimir, Kosicki Michael, Bassett Andrew R, Harding Heather, Galanty Yaron, Muñoz-Martínez Francisco, Metzakopian Emmanouil, Jackson Stephen P, Parts Leopold
Wellcome Sanger Institute, Hinxton, UK.
Cambridge Institute of Medical Research, University of Cambridge, Cambridge, UK.
Nat Biotechnol. 2018 Nov 27. doi: 10.1038/nbt.4317.
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 CRISPR-Cas9 reagents. In total, we gathered data for >10 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.
由CRISPR-Cas9产生的双链断裂经细胞修复产生的DNA突变决定了其表型效应。已知突变结果并非随机产生，而是取决于靶向位置的DNA序列。在此，我们通过测量合成构建体中40000多个导向RNA（gRNA）产生的编辑，系统地研究了侧翼DNA序列对修复结果的影响。我们在一系列遗传背景下并使用替代的CRISPR-Cas9试剂进行了实验。总共，我们收集了超过10种突变结果的数据。大多数可重复的突变是单个碱基的插入、短缺失或更长的微同源性介导的缺失。每个gRNA对特定结果都有个体细胞系依赖性偏向。我们揭示了所产生突变的序列决定因素，并利用这些因素得出Cas9编辑结果的预测指标。对序列修复的深入理解将有助于更好地设计基因编辑实验。
