CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library

• Published in: Cell stem cell Vol. 17; no. 2; pp. 221 - 232
• Main Authors: Zhong, Cuiqing, Yin, Qi, Xie, Zhenfei, Bai, Meizhu, Dong, Rui, Tang, Wei, Xing, Yu-Hang, Zhang, Hongling, Yang, Suming, Chen, Ling-Ling, Bartolomei, Marisa S., Ferguson-Smith, Anne, Li, Dangsheng, Yang, Li, Wu, Yuxuan, Li, Jinsong
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.08.2015
• Subjects: Alleles; Animals; Base Sequence; Cloning, Organism; CRISPR-Cas Systems - genetics; DNA Methylation - genetics; Gene Library; Genetic Testing; Haploidy; Heterozygote; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Molecular Sequence Data; Mouse Embryonic Stem Cells - metabolism; RNA, Guide, CRISPR-Cas Systems - metabolism
• ISSN: 1934-5909; 1875-9777
• DOI: 10.1016/j.stem.2015.06.005

Mouse androgenetic haploid embryonic stem cells (AG-haESCs) can support full-term development of semi-cloned (SC) embryos upon injection into MII oocytes and thus have potential applications in genetic modifications. However, the very low birth rate of SC pups limits practical use of this approach. Here, we show that AG-haESCs carrying deletions in the DMRs (differentially DNA methylated regions) controlling two paternally repressed imprinted genes, H19 and Gtl2, can efficiently support the generation of SC pups. Genetic manipulation of these DKO-AG-haESCs in vitro using CRISPR-Cas9 can produce SC mice carrying multiple modifications with high efficiency. Moreover, transfection of DKO-AG-haESCs with a constitutively expressed sgRNA library and Cas9 allows functional mutagenic screening. DKO-AG-haESCs are therefore an effective tool for the introduction of organism-wide mutations in mice in a single generation. [Display omitted] •Misexpression of imprinted genes hinders application of haploid mouse ESCs•The mutation of two paternally imprinted genes improves semi-cloning efficiency•Genetic manipulation using CRISPR-Cas9 efficiently leads to mutant mice•Introduction of a CRISPR-Cas9-based library enables mutagenic screening Li and colleagues show that the combined application of altered expression of two imprinted genes and CRISPR-Cas9-based genome editing allows the efficient and stable generation of gene-modified semi-cloned mice from androgenetic haploid embryonic stem cells. This approach has potential for mutagenesis and screening.