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

Abstract

Abstract 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.

Keywords

• CRISPR-Cas9
• Nonobstructive azoospermia
• Spermatogonia stem cells