CRISPR/Cas9 applied to editing nonhuman primate cells and embryos Rb1 suppressor gene

Objective To explore the editing efficiency of CRISPR/Cas9 gene editing system for cynomolgus monkey cells and embryos retinoblastoma tumor suppressor gene (Rb1) in order to further establish retinoblastoma model. Methods Rb1 mutation database (http://rb1-lsdb.d-lohmann.de) was referred to determine exon 8 which was the main exon of Rb1 that related to the incidence and development of retinoblastoma tumor; DNAman was applied to detect whether there was a SNP in the Rb1 gene between human and macaque monkey; Then we use http://crispr.cos.uni-heidelberg.de/index.html to design the sgRNA sequence; The CRISPR/Cas9 gene editing system was performed to detect the knock-out efficiency by transfecting RNP (sgRNA and Cas9 nuclease mixture) into the cells; Enzyme digestion experiment was used to detect the type of mutations, and monoclonal gene analysis to determine the specific knock-out efficiency. Further microinjection technique was performed to inject RNP into singlecell embryos to detect embryonic Rb1 gene editing efficiency. Results The genome of the RNP-transfected cynomolgus monkey fi broblast cells were extracted, 75% of the cells were found to have gene mutations, and the type of gene editing varied, with basedeleted mutations being dominant; 4-cell stage embryo was obtained and the detection of a single blastomere genome sequence revealed that the mutant embryos accounted for 36.4% of all embryos, of which the homozygous mutation ratio was 18.2% and the chimeric mutation ratio was 18.2%. Twelve off-target sites were predicted using software, and sequencing was performed. As a result, no off-target was found. Conclusion In non-human primate cells and embryos, the CRISPR/Cas9 gene editing system is effective for Rb1 editing of cynomolgus monkeys, and it has a higher knock-out efficiency, which means using gene editing methods to establish a retinoblastoma model can be achieved later. As an effective gene editing tool, the editing efficiency of Rb1 gene and the rate of embryonic blastocyst in the cynomolgus monkey embryo provide a reference for the establishment of retinoblastoma model.