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Highlights • CRISPR/Cas9 induces random deletions in the target region of GABRR2 gene. • Both big and small indels can lead to unexpected high probability of exon truncation/skipping and exon recruitment. • It is the first observation of exon recruitment by CRISPR/Cas9-mediated GABRR2 gene editing. Abstract 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. [ABSTRACT FROM AUTHOR]

Abstract: Recent discovery of biological function of endothelial cell-specific chemotaxic regulator (ECSCR), previously known as endothelial cell-specific molecule 2 (ECSM2), in modulating endothelial cell migration, apoptosis, and angiogenesis, has made it an attractive molecule in vascular research. Thus, identification of splice variants of ECSCR could provide new strategies for better understanding its roles in health and disease. In this study, we performed a series of blast searches on the human EST database with known ECSCR cDNA sequence (Variant 1), and identified additional three splice variants (Variants 2–4). When examining the ECSCR gene in the human genome assemblies, we found a large unknown region between Exons 9 and 11. By PCR amplification and sequencing, we partially mapped Exon 10 within this previously unknown region of the ECSCR gene. Taken together, in addition to previously reported human ECSCR, we identified three novel full-length splice variants potentially encoding different protein isoforms. We further defined a total of twelve exons and nearly all exon–intron boundaries of the gene, of which only eight are annotated in current public databases. Our work provides new information on gene structure and alternative splicing of the human ECSCR, which may imply its functional complexity. This undoubtedly opens new opportunities for future investigation of the biological and pathological significance of these ECSCR splice variants. [Copyright &y& Elsevier]