title: Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells.
abstract: Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc(-/-)) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [3] College of Life Science, China West Normal University, Nanchong, Sichuan 637002, China.
authors.forename: Hai
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Biodynamic Optical Imaging Center, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Fan
authors.affiliation: 1] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Man
authors.initials: M
authors.lastname: Zhang
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Zhenfei
authors.initials: Z
authors.lastname: Xie
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [3] School of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China.
authors.forename: Meizhu
authors.initials: M
authors.lastname: Bai
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yin
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Liang
authors.affiliation: Animal Core Facility, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Jiaoyang
authors.initials: J
authors.lastname: Liao
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Chikai
authors.initials: C
authors.lastname: Zhou
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Wujuan
authors.initials: W
authors.lastname: Liu
authors.affiliation: Biodynamic Optical Imaging Center, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhu
authors.affiliation: Biodynamic Optical Imaging Center, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Hongshan
authors.initials: H
authors.lastname: Guo
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Pan
authors.affiliation: College of Life Science, China West Normal University, Nanchong, Sichuan 637002, China.
authors.forename: Chunlian
authors.initials: C
authors.lastname: Wu
authors.affiliation: National Population and Family Planning Committee, Key Laboratory of Contraceptive Drugs and Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Shi
authors.affiliation: 1] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Ligang
authors.initials: L
authors.lastname: Wu
authors.affiliation: Biodynamic Optical Imaging Center, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Fuchou
authors.initials: F
authors.lastname: Tang
authors.affiliation: 1] Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2] Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [3] School of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China.
authors.forename: Jinsong
authors.initials: J
authors.lastname: Li
mesh_terms: D053687:Adult Stem Cells / Q000378:metabolism* / Q000637:transplantation*; D000818:Animals; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D059766:DNA End-Joining Repair; D005260:Female; D018773:Genes, erbB-1; D015316:Genetic Therapy; D007248:Infertility, Male / Q000235:genetics* / Q000628:therapy*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016296:Mutagenesis; D013091:Spermatogenesis; D019076:Transgenes; D038222:gamma-Crystallins / Q000235:genetics
chemical_list: D038222:gamma-Crystallins; C554179:gammaC-crystallin protein, mouse