The CRISPR-Cas9 system has been employed to generate mutant alleles in a range of different organisms. However, so far there have not been reports of use of this system for efficient correction of a genetic disease. Here we show that mice with a dominant mutation in Crygc gene that causes cataracts could be rescued by coinjection into zygotes of Cas9 mRNA and a single-guide RNA (sgRNA) targeting the mutant allele. Correction occurred via homology-directed repair (HDR) based on an exogenously supplied oligonucleotide or the endogenous WT allele, with only rare evidence of off-target modifications. The resulting mice were fertile and able to transmit the corrected allele to their progeny. Thus, our study provides proof of principle for use of the CRISPR-Cas9 system to correct genetic disease.

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...