Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells
- Stem Cell Reports. 2016 Sep 13;7(3):496-507. doi: 10.1016/j.stemcr.2016.07.021.
- 1. Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
- 2. Department of Anatomy and Neurobiology, the Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, China.
- 3. College of Animal Science and Zhejiang University School of Medicine, Zhejiang University, Hangzhou 310058, China.
- 4. Department of Anatomy and Neurobiology, the Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, China. Electronic address: [email protected].
- 5. Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China; Tongji University Advanced Institute of Translational Medicine, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, China. Electronic address: [email protected].
- 6. Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China; Tongji University Advanced Institute of Translational Medicine, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, China; The Collaborative Innovation Center for Brain Science, Tongji University, Shanghai 200092, China. Electronic address: [email protected].
Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeeded in targeting all genes of interest with high biallelic targeting efficiencies. More importantly, during paired-KO, the cleaved DNA was repaired mostly through direct end joining without insertions/deletions (precise ligation), and thus makes the lesion product predictable. The paired-KO remained highly efficient for one-step targeting of multiple genes and was also efficient for targeting of MicroRNA, while for long non-coding RNA over 8 kb, cleavage of a short fragment of the core promoter region was sufficient to eradicate downstream gene transcription. This work suggests that the paired-KO strategy is a simple and robust system for loss-of-function studies for both coding and non-coding genes in hPSCs.