CRISPR/Cas9-mediated editing of GABRR2 gene in RGC-5 cells induces random exon deletion, exon splicing and new exon recruitment

CRISPR/Cas9 and its variations provide an efficient tool for targeted genome editing. CRISPR/Cas9-based genome editing can induce mutations in genome that has high probability to cause exon truncation or deletion. However, screening mutations in diploid cells is difficult because of two copies of chromosome. It is an important task for determining genotypes in diploid cells subjected to editing before using these cells in gene function study. In this study, we applied CRISPR/Cas9 to edit the GABRR2 gene in mouse retinal ganglion cells to study what exactly happened in two alleles and what real mRNA isoforms formed in diploid cells. A single sgRNA was employed to generate double-strand DNA breaks. PCR sequencing was used for single clone validation in diploid cells subjected to editing. The indels and the corresponding effects at the target locus were further studied at genomic and RNA levels. We observed that CRISPR/Cas9 induces random deletions in the target region of GABRR2 gene, and both big and small indels can lead to unexpected high probability of exon truncation/skipping. In addition, random deletions in genomic region recruited introns to generate new “exon”. It is the first observation of exon recruitment by CRISPR/Cas9-mediated GABRR2 gene editing. The observations may offer a reference for the future gene splicing study.