Long-read sequence analysis of MMEJ-mediated CRISPR genome editing in mouse ES cells
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 unexpected 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 showed a high frequency of bands of 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. Our study indicates that careful quality control of genome-edited clones is needed to exclude complex, unexpected, on-target vector insertion.