When used to edit genomes, Cas9 nucleases produce targeted double-strand breaks in DNA. Subsequent DNA-repair pathways can induce large genomic deletions (larger than 100 bp), which constrains the applicability of genome editing. Here we show that Cas9-mediated double-strand breaks induce large deletions at varying frequencies in cancer cell lines, human embryonic stem cells and human primary T cells, and that most deletions are produced by two repair pathways: end resection and DNA-polymerase theta-mediated end joining. These findings required the optimization of long-range amplicon sequencing, the development of a k-mer alignment algorithm for the simultaneous analysis of large DNA deletions and small DNA alterations, and the use of CRISPR-interference screening. Despite leveraging mutated Cas9 nickases that produce single-strand breaks, base editors and prime editors also generated large deletions, yet at approximately 20-fold lower frequency than Cas9. We provide strategies for the mitigation of such deletions.

当用于编辑基因组时，Cas9 核酸酶会在 DNA 中产生靶向双链断裂。随后的 DNA 修复途径可诱导大基因组缺失（大于 100 bp），这限制了基因组编辑的适用性。在这里，我们表明 Cas9 介导的双链断裂在癌细胞系、人胚胎干细胞和人原代 T 细胞中以不同频率诱导大缺失，并且大多数缺失是由两种修复途径产生的：末端切除和 DNA 聚合酶 θ 介导的末端连接。这些发现需要优化长片段扩增子测序，开发用于同时分析大 DNA 缺失和小 DNA 改变的 k-mer 比对算法，以及使用 CRISPR 干扰筛选。尽管利用产生单链断裂的突变 Cas9 切口酶，但碱基编辑器和引物编辑器也产生了大缺失，但频率比 Cas9 低约 20 倍。我们提供减少此类删除的策略。