Strategies to edit paralogous genes with CRISPR/Cas9

Abstract

The purpose of this study is to develop the strategies of CRISPR/Cas9 application to improve fidelity and specificity of this platform. Here we use a model system, which includes target gene and a paralogue - potential aim for off-target double-strand break induction. The study was carried on using Brattleboro rats embryonic fibroblasts which are homozygous for a mutation in arginine-vasopressin gene (target). The potential off-target gene is oxytocin gene: its DNA sequence is almost identical to that of arginine-vasopressin gene. To prevent off-target effect we designed several strategies, which were further used on Brattleboro rats embryonic fibroblasts. Here we show, that these strategies allowed us to generate double-strand breaks in arginine-vasopressin gene without any off-target effects in oxytocin gene. The endonuclease restriction assay shows that we have modified arginine-vasopressin gene while using both CRISPR/Cas9 and single-stranded oligonucleotides as a donor for homologous recombination. At last, if we consider Brattleboro rats as a model of monogenic disease the strategies designed could be translated in human therapeutic genome editing studies.