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Correcting Genetic Disease in Mice through CRISPR-Cas9-mediated Gene Editing in Spermatogonial Stem Cells
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The CRISPR-Cas9 system from bacteria has been recently employed in gene therapy. Two studies, one from Dr. LI Jinsong’s lab (Cell Stem Cell, 2013, 13, 659) and the other from Dr. Olson’s lab (Science, 2014, 345, 1184) reported that the CRISPR-Cas9 system, after being injected into zygotes, could correct disease-causing mutations in mice. Nevertheless, direct injection of CRISPR-Cas9 system into zygotes could not produce healthy progeny at an efficiency of 100% and could potentially generate off-target modifications. To circumvent these problems, one possible strategy is that successful gene editing in spermatogonia stem cells (SSCs) that contain a disease-causing mutation using the CRISPR-Cas9 system should allow for the selection of single SSCs that carry the desired gene modification but without any other unwanted genomic changes and that such SSC lines could then be used to produce healthy offspring at 100% efficiency. Recently, a study from researchers of Shanghai Institutes for Biological Sciences (SIBS) and Peking University provided proof of principle for use of the CRISPR-Cas9 to correct genetic disease through SSCs.

To test the feasibility of the strategy of CRISPR-Cas9-mediated genetic repair in SSCs, WU Yuxuan, ZHOU Hai, FAN Xiaoying, ZHANG Ying, ZHANG Man and their colleagues, led by Drs. LI Jinsong and Wu Ligang at Institute of Biochemistry and Cell Biology, SIBS, CAS and Dr. TANG Fuchou at Peking University, generated a SSC line from homozygous cataract male (termed Crygc-/--SSCs). After electroporation of CRISPR-Cas9 or CRISP-Cas9 plus exogenous wildetype single-stranded DNA oligonucleotides into Crygc-/--SSCs, they derived multiple SSC lines that carried corrected Crygc gene via NHEJ (non-homologous end joining) or HDR (homology-directed repair)-mediated gene editing. They analyzed off-target effects in these cells by sequencing predicted potential off-targets loci and by whole-genome sequencing. They further analyzed the imprinting state of these cells by bisulfite analysis of specific imprinting genes and by whole-genome bisulfite sequencing. Fertilization using spermatids derived from these corrected SSC lines gave rise to offspring with the corrected phenotype at an efficiency of 100%.

This work entitled “Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells” was published in Cell Research on Dec 5, 2014.

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

LI Jinsong
Institute of Biochemistry and Cell Biology, SIBS, CAS,
Shanghai 200031, China
Phone: +86-21-54921422
E-mail: jsli@sibcb.ac.cn

Fig.Diagram for the correction of a genetic disease in mouse through CRISPR-Cas9-mediated gene editing in SSCs.
(Image Provided by Dr. LI Jinsong)


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