CRISPR-Cas9-Mediated Gene Editing in Mouse Spermatogonial Stem Cells
Precise genome editing is a powerful tool for analysis of gene function. However, in spermatogonial stem cells (SSCs), this still remains a big challenge mainly due to low efficiency and complexity of currently available gene editing techniques. The CRISPR-Cas9 system from bacteria has been applied to modifying genome in different species at a very high efficiency and specificity. Here we describe CRISPR-Cas9-mediated gene editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in SSCs. This protocol provides guidelines for derivation of SSCs, nucleofection of SSCs with the CRISPR-Cas9 system, transplantation of the gene-modified SSCs into the recipient testes, and production of mice using transplanted SSC-derived round spermatids.
Key wordsCRISPR-Cas9 Spermatogonial stem cells Transplantation Nucleofection Round spermatid injection (ROSI) Embryo transfer
This study was supported by grants from the Ministry of Science and Technology of China (2014CB964803, 2015AA020307 and 2013CB967103), the National Natural Science Foundation of China (31530048 and 81672117), the Chinese Academy of Sciences (XDB19010204), and the Shanghai Municipal Commission for Science and Technology (16JC1420500).
- 24.Nagy N, Gertsenstein M, Vintersten K, Behringer R (2002) Manipulating the mouse embryo-a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar