Microhomology-mediated endjoining repair mechanism enables rapid and effective indel generations in stem cells

Abstract

Functional characterization of gene(s) using a transgene approach in a human cell line or in an animal model generally poses limitations due to persistent transgene overexpression. Conversely, the CRISPR/Cas9 geneediting technology enables precise variant(s) introduction in a gene, thus facilitating accurate characterization in human iPSC-derived target cell/tissue. Such editing is generally mediated by non-homologous end joining, the predominant and error-prone double-strand break repair mechanism which mostly results in gene knockout due to indel(s) generation. However, in most cases the best in silico predicted sgRNAs fail to generate indels especially in iPSCs, encouraging a revisit of DNA damage repair principles. Microhomology-mediated end joining (MMEJ) is another error-prone repair mechanism which relies on exposed microhomologous sequences flanking the broken ends to fix double-strand breaks. Therefore, sgRNAs targeting the exonic region encompassing di- or tri-nucleotide repeats along with non-repeat exonic region as control, in RIC3, a gene of our interest, were designed to generate effective indel(s) exploiting the MMEJ DSB repair mechanism. iPSCs were co-transfected with eCas9+EGFP and sgRNA+puromycin plasmids and positive clones enriched by transient puromycin selection. Multiple deletion lines adjacent to microhomologous (repeat) region and several lines heterozygous with only 1 bp insertion for the non-repeat region were obtained, and confirmed by Sanger sequencing. These findings suggest that (i) designing sgRNAs from different exonic regions with microrepeats and (ii) MMEJ combined with a rapid and less expensive method of using antibiotic screening of edited lines without cumbersome cell sorting may be effective strategies for indel(s) generation in stem cells.