In 2012, a research group led by Profs. LI Jinsong and XU Guoliang published a Cell paper reporting the creation of androgenetic haploid embryonic stem cells (AG-haESCs) that can support full-term embryonic development upon injection into MII oocytes, leading to the generation of semi-cloned (SC) mice (Cell, 2012, 149: 605-617.). This study represented an important conceptual advance by the creation of a novel, renewable/culturable form of fertilization agent. Nevertheless, one major drawback of the previous work is the frequently observed aberrant development of AG-haESC-derived embryos and the very low birth rate of healthy SC mice (around 2% of total SC embryos), which restricts the applications of AG-haESCs.  

On July 9th, a team of researchers from Shanghai Institutes for Biological Sciences led by LI Jinsong, WU Yuxuan of the Institute of Biochemistry and Cell Biology, and YANG Li of CAS-MPG Partner Institute for Computational Biology, have shown that AG-haESCs carrying deletions in the DMRs (differentially DNA methylated regions) controlling two paternally repressed imprinted genes, H19 and Gtl2, designated as DKO-AG-haESCs, can efficiently support the generation of SC pups at a rate of 20%.  

Moreover, the team demonstrated that manipulation of multiple genes is feasible in DKO-AG-haESCs, leading to the generation of SC mice carrying multiple genetic traits at an appropriate efficiency.  

Importantly, they further demonstrated that by combination of CRISPR-Cas9 library and DKO-AG-haESCs, SC mice carrying different mutant genes can be efficiently generated in one step, thus enabling functional mutagenic screening at organism level in mice, which can be done currently in lower organisms, such as yeast and C. elegans.  

The researchers proposed that this new technology is feasible for medium-scale targeted screening at organism level, especially for developmental phenotypes, using the appropriate sgRNA libraries targeting preselected candidate genes.   

This work entitled “CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library” was published online in Cell Stem Cell on July 9th. 

This study was supported by the grants from the Chinese Academy of Sciences (the Strategic Priority Research Program), the Ministry of Science and Technology of China, the National Natural Science Foundation of China and the Science and Technology Commission of Shanghai Municipality.  

CONTACT:
LI Jinsong, Principal Investigator
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
Shanghai 200031, P. R. China
E-mail: jsli@sibcb.ac.cn 

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. (Image by Prof. LI Jinsong`s group)