Year: 2016
Type: article
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.
Source: Genes and Cells
DOI: 10.23868/gc120594
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4403481780