Restore natural fertility of Kit(w)/Kit(wv) mouse with nonobstructive azoospermia through gene editing on SSCs mediated by CRISPR-Cas9

BACKGROUND: Male infertility is a serious social problem in modern society. Nonobstructive azoospermia (NOA) caused by germ cell gene defects is an important reason for male infertility, but effective clinical treatment for this disease has not been established. METHODS: We choose Kit(w)/Kit(wv) mouse as a research model and try to develop a new treatment strategy and “cure” its infertility. Mutant spermatogonial stem cells (SSCs) were isolated from one single unilateral testis of a 14-day-old Kit(w)/Kit(wv) mouse and propagated in vitro. The C to T point mutation on Kit(wv) site of these SSCs was corrected through CRISPR-Cas9-mediated homology-directed repair (HDR) in vitro. Then, the repaired SSCs were screened out, proliferated, and transplanted into the remaining testis, and complete spermatogenesis was established in the recipient testis. RESULTS: Healthy offsprings with wild type Kit gene or Kit(w) mutation were obtained through natural mating 4 months after SSC transplantation. CONCLUSION: In this study, we established an effective new treatment strategy for NOA caused by germ cell gene defects through a combination of SSC isolation, CRISPR-Cas9-mediated gene editing, and SSC transplantation, which brought hope for these NOA patients to restore their natural fertility.