Curing genetic mutations for the next generation: CRISPR/Cas9-mediated gene correction in mouse spermatogonial stem cells

Abstract

The CRISPR/Cas system is a very powerful and versatile tool for genomic manipulation. A recent study published in Cell Research by Wu et al. [1] reported a Cas9-mediated strategy to correct a disease-causing mutation in the mouse gammaC-crystallin(Crygc) locus in spermatogonial stem cells (SSCs) to cure the resulting cataracts disease in the offspring with 100 % efficiency. Importantly, no off-target mutations were identified through whole-genome sequencing. This study is a perfect combination of three different high biological technologies, SSCs manipulation, CRISPR/ Cas gene editing and round spermatid injection (ROSI), shedding light on clinical applications for repair of genetic mutations in the progeny. Many genetic disorders are caused by DNA mutation(s) which is usually inherited from a parental genome and potentially passes to the next generation. Although some symptoms of certain genetic diseases are alleviated with medical or surgical treatments, the disease is hard to cure due to limitations of available approaches to manipulate the human genome efficiently. In recent years, with the emergence of programmable nucleases, scientists are able to edit specific genomic sites by creating DNA doublestrand breaks (DSBs) which stimulate DNA repair either through nonhomologous end joining (NHEJ) or homologydirected repair (HDR) pathways depending on the presence of the repair template [2]. Compared with zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system is more popular due to its simplicity, efficiency and flexibility [3]. The CRISPR/Cas system is derived from the adaptive immune system in bacteria and archaea [4]. It consists of the endonuclease Cas9 (from S. pyogenes) and a customized single-guide RNA (sgRNA) that leads the Cas9 to the DNA target following base-paring rules [4]. With the CRISPR/ Cas system, researchers have demonstrated quick generation of gene-edited model organisms [5, 6] or correction of genetic mutations in animals [7, 8]. Although both somatic and one-cell embryo injection of CRISPR/Cas-mediated gene repair alleviates disease symptoms, these studies neither could cure the disease nor avoid off-target digestion of Cas9 endonuclease. More importantly, the mutations were still transmissable to the next generation. Spermatogonial stem cells (SSCs) are self-renewing male germ line stem cells that are responsible for producing numerous spermatozoa and transmitting genetic information through fertilization. The techniques of in vitro culture and proliferation of SSCs from different species including humans have been well developed [9, 10]. Recently, by taking advantages of SSC manipulation, the CRISPR/Cas system and ROSI, Wu et al. took the lead in gene editing in mouse SSCs and the generation of genetically modified animal models. Moreover, they also used the strategy to correct a genetic disease in the new born pups with 100 % efficiency [1]. To test the feasibility, Wu et al. first disrupted an eGFP transgene in the SSCs in vitro by electroporation of plasmids expressing Cas9 and sgRNA targeting eGFP. The SSCs derived from actin-eGFP transgenic male mice were stably transduced with an mRFP marker to facilitate tracing. Two weeks after transfection of Cas9–sgRNA plasmids, the SSCs expressing mRFP alone were sorted. The sorted SSCs were transplanted into the seminiferous tubules of busulfan-treated males. Round spermatids from the recipient males were isolated and injected into wildtype oocytes to produce mice. After sequencing, half of the D. Li (&) Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China e-mail: dlli@bio.ecnu.edu.cn