Department of Physiology II, Nara Medical University, Kashihara, Nara, 634-8521, Japan.
Sci Rep. 2023 Jul 19;13(1):11652. doi: 10.1038/s41598-023-38397-y.
CRISPR genome editing is a powerful tool for elucidating biological functions. To modify the genome as intended, it is essential to understand the various modes of recombination that can occur. In this study, we report complex vector insertions that were identified during the generation of conditional alleles by CRISPR editing using microhomology-mediated end joining (MMEJ). The targeting vector contained two loxP sequences and flanking 40-bp microhomologies. The genomic regions corresponding to the loxP sequences were cleaved with Cas9 in mouse embryonic stem cells. PCR screening for targeted recombination revealed a high frequency of bands of a larger size than expected. Nanopore sequencing of these bands revealed complex vector insertions mediated not only by MMEJ but also by non-homologous end joining and homologous recombination in at least 17% of the clones. A new band appeared upon improving the PCR conditions, suggesting the presence of unintentionally modified alleles that escape standard PCR screening. This prompted us to characterize the recombination of each allele of the genome-edited clones using heterozygous single nucleotide polymorphisms, leading to confirmation of the presence of homozygous alleles. Our study indicates that careful quality control of genome-edited clones is needed to exclude complex, unintended, on-target vector insertion.
CRISPR 基因组编辑是阐明生物学功能的强大工具。为了按预期修改基因组，了解可能发生的各种重组模式至关重要。在这项研究中，我们报告了通过使用微同源介导末端连接 (MMEJ) 的 CRISPR 编辑生成条件等位基因时鉴定出的复杂载体插入。靶向载体包含两个 loxP 序列和侧翼 40bp 微同源序列。loxP 序列对应的基因组区域在小鼠胚胎干细胞中被 Cas9 切割。针对靶向重组的 PCR 筛选显示，比预期大的条带的频率很高。对这些条带进行纳米孔测序表明，复杂的载体插入不仅由 MMEJ 介导，而且在至少 17%的克隆中由非同源末端连接和同源重组介导。在改进 PCR 条件后出现了一个新的条带，表明存在逃避标准 PCR 筛选的意外修饰等位基因。这促使我们使用杂合单核苷酸多态性来表征基因组编辑克隆的每个等位基因的重组，从而证实存在纯合等位基因。我们的研究表明，需要仔细的基因组编辑克隆质量控制来排除复杂的、非预期的、靶向载体插入。
