Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

• DOI: 10.1038/srep13348
• Publication Date: 2015-08-21T09:18:03Z
• Authors: Xianlong Wang; Jinwei Zhou; Chunwei Cao; Jiaojiao Huang; Tang Hai; Yanfang Wang; Qiantao Zheng; Hongyong Zhang; Guosong Qin; Xiangnan Miao; Hongmei Wang; Suizhong Cao; Qi Zhou; Jianguo Zhao
• Keywords: Guide RNA; Genome Engineering; Subgenomic mRNA; Gene targeting

Abstract Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, uncertainties caused wide variations sgRNA activity impede utility this system generating genetically modified pigs. Here, we described single blastocyst genotyping provide simple and rapid solution evaluate compare efficiency at inducing indel mutations for given gene locus. Assessment mutagenesis efficiencies achieved within 10 days from design sgRNA. The most effective selected successfully used induce insertion through homology-directed repair frequency exceeding 13%. Additionally, highly efficient deletion via confirmed pig fibroblast cells, could serve as donor cells somatic cell nuclear transfer. We further showed that direct cytoplasmic injection Cas9 mRNA favorable into zygotes biallelic knockout piglets an up 100%. Thus, our method considerably reduces expands practical possibilities CRISPR/Cas9-mediated genome