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. Keywords: CRISPR-Cas9; spermatogonial stem cell; gene therapy Cell Research (2015) IntroductionSpermatogonial stem cells (SSCs) can self-renew and undergo spermatogenesis, leading to the production of numerous spermatozoa, which transmit the genetic information to the next generation [1,2]. SSCs from different species can be maintained in vitro for long periods of time in medium supplemented with glial cell line-derived neurotrophic factor (GDNF) [3][4][5][6][7]. Meanwhile, after transplantation into the testes of an infertile male, cultured SSCs can re-establish spermatogenesis and restore fertility [1,8,9]. As genetic manipulation of SSCs and the subsequent transplantation allow one to select for desired genetic modifications, these techniques hold great promise in producing gene-modified animal models and particularly in treating genetic diseases with the potential of generating healthy progeny at 100% efficiency [1,10]. However, so far there have been very limited reports of using these techniques for efficient production of gene-modified animals [11,12], and their use in genetic disease correction has not yet been reported, partially due to complexity and low efficiency of currently available genetic editing techniques.Recently, the CRISPR-Cas9 system from bacteria has enabled rapid genome editing in different species at a very high efficiency and specificity [13][14][15][16][17]. CRIS-PR-Cas9-mediated genome editing requires only a short single-guide RNA (sgRNA) to guide site-specific DNA recogni...

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.