CRISPR-Cas9 genome editing has promising therapeutic potential for genetic diseases and cancers, but safety could be a concern. Here we use whole genomic analysis by 10x linked-read sequencing and optical genome mapping to interrogate the genome integrity after editing and in comparison to four parental cell lines. In addition to the previously reported large structural variants at on-target sites, we identify heretofore unexpected large chromosomal deletions (91.2 and 136 Kb) at atypical non-homologous off-target sites without sequence similarity to the sgRNA in two edited lines. The observed large structural variants induced by CRISPR-Cas9 editing in dividing cells may result in pathogenic consequences and thus limit the usefulness of the CRISPR-Cas9 editing system for disease modeling and gene therapy. In this work, our whole genomic analysis may provide a valuable strategy to ensure genome integrity after genomic editing to minimize the risk of unintended effects in research and clinical applications.

CRISPR-Cas9基因组编辑对于遗传疾病和癌症具有广阔的治疗潜力，但安全性可能是一个问题。在这里，我们通过 10 倍链读测序和光学基因组图谱进行全基因组分析，以询问编辑后的基因组完整性，并与四种亲本细胞系进行比较。除了之前报道的在靶位点的大结构变异之外，我们还在非典型非同源脱靶位点鉴定了迄今为止意想不到的大染色体缺失（91.2和136 Kb），与两个编辑品系中的sgRNA没有序列相似性。在分裂细胞中观察到的 CRISPR-Cas9 编辑诱导的大结构变异可能会导致致病后果，从而限制 CRISPR-Cas9 编辑系统在疾病建模和基因治疗中的用途。在这项工作中，我们的全基因组分析可能提供一种有价值的策略，以确保基因组编辑后基因组的完整性，从而最大限度地减少研究和临床应用中意外影响的风险。