Chao, Tianzhu Liu, Zhuangzhuang Zhang, Yu Zhang, Lichen Huang, Rong He, Le Gu, Yanrong Chen, Zhijun Zheng, Qianqian Shi, Lijin Zheng, Wenping Qi, Xinhui Kong, Eryan Zhang, Zhongjian Lawrence, Toby Liang, Yinming Lu, Liaoxun Presentation_1_Precise and Rapid Validation of Candidate Gene by Allele Specific Knockout With CRISPR/Cas9 in Wild Mice.PPTX <p>It is a tempting goal to identify causative genes underlying phenotypic differences among inbred strains of mice, which is a huge reservoir of genetic resources to understand mammalian pathophysiology. In particular, the wild-derived mouse strains harbor enormous genetic variations that have been acquired during evolutionary divergence over 100s of 1000s of years. However, validating the genetic variation in non-classical strains was extremely difficult, until the advent of CRISPR/Cas9 genome editing tools. In this study, we first describe a T cell phenotype in both wild-derived PWD/PhJ parental mice and F1 hybrids, from a cross to C57BL/6 (B6) mice, and we isolate a genetic locus on Chr2, using linkage mapping and chromosome substitution mice. Importantly, we validate the identification of the functional gene controlling this T cell phenotype, Cd44, by allele specific knockout of the PWD copy, leaving the B6 copy completely intact. Our experiments using F1 mice with a dominant phenotype, allowed rapid validation of candidate genes by designing sgRNA PAM sequences that only target the DNA of the PWD genome. We obtained 10 animals derived from B6 eggs fertilized with PWD sperm cells which were subjected to microinjection of CRISPR/Cas9 gene targeting machinery. In the newborns of F1 hybrids, 80% (n = 10) had allele specific knockout of the candidate gene Cd44 of PWD origin, and no mice showed mistargeting of the B6 copy. In the resultant allele-specific knockout F1 mice, we observe full recovery of T cell phenotype. Therefore, our study provided a precise and rapid approach to functionally validate genes that could facilitate gene discovery in classic mouse genetics. More importantly, as we succeeded in genetic manipulation of mice, allele specific knockout could provide the possibility to inactivate disease alleles while keeping the normal allele of the gene intact in human cells.</p> CD44;allele specific knockout;CRISPR/Cas9;functional genomics;wild mice 2019-02-19
    https://frontiersin.figshare.com/articles/presentation/Presentation_1_Precise_and_Rapid_Validation_of_Candidate_Gene_by_Allele_Specific_Knockout_With_CRISPR_Cas9_in_Wild_Mice_PPTX/7738877
10.3389/fgene.2019.00124.s002