Homology-independent targeted integration in vivo restores Cldn11 deficiency in mouse Sertoli cells and spermatogenesis

Abstract Defective testicular Sertoli cell (SC) function may be an underlying cause of male infertility/subfertility; however, in vivo gene therapy for nonobstructive azoospermia (NOA) caused by SCs has never been attempted. In this study, a CRISPR/Cas9-based homology-independent targeted integration (HITI) gene editing strategy was used to integrate an ∼3.7 kb DNA fragment containing a SC-specific enhancer/promoter and a region comprising three Cldn11 exons into the SCs of Cldn11-deficient mice. After the viral plasmids carried by recombinant adeno-associated virus serotype 1 (rAAV1) were delivered to SCs via testicular tubular injection, the phenotypes of blood‒testis barrier loss, spermatogenesis blockage, and infertility in the mice were successfully rescued for 6 months, and first-generation offspring were produced using the sperm of the rescued mice. Our results suggest that despite the lack of proliferation and low division capacity of adult testicular SCs, the introduction of targeted strategies such as HITI could enable in vivo gene editing as a possible treatment for infertility caused by Sertoli cell defects. Competing Interest Statement The authors have declared no competing interest.