Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells
Qiupeng Zheng,
Xiaohong Cai,
Meng How Tan,
Steven Schaffert,
Christopher P. Arnold,
Xue Gong,
Chang-Zheng Chen,
Shenglin Huang
Affiliations
Qiupeng Zheng
1Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, and Department of Oncology, Shanghai Medical School, Fudan University, Shanghai, China
Xiaohong Cai
2Baxter Laboratory for Stem Cell Biology, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA
Meng How Tan
3School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore
Steven Schaffert
2Baxter Laboratory for Stem Cell Biology, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA
Christopher P. Arnold
2Baxter Laboratory for Stem Cell Biology, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA
Xue Gong
2Baxter Laboratory for Stem Cell Biology, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA
Chang-Zheng Chen
2Baxter Laboratory for Stem Cell Biology, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA
Shenglin Huang
1Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, and Department of Oncology, Shanghai Medical School, Fudan University, Shanghai, China
The prokaryotic type II CRISPR/Cas9 system has been adapted to perform targeted genome editing in cells and model organisms. Here, we describe targeted gene deletion and replacement in human cells via the CRISPR/Cas9 system using two guide RNAs. The system effectively generated targeted deletions of varied length, regardless of the transcriptional status of the target gene. It is notable that targeted gene deletions generated via CRISPR/Cas9 and two guide RNAs resulted in the formation of correct junctions at high efficiency. Moreover, in the presence of a homology repair donor, the CRISPR/Cas9 system could guide precise gene replacement. Our results illustrate that the CRISPR/Cas9 system can be used to precisely and effectively generate targeted deletions or gene replacement in human cells, which will facilitate characterization of functional domains in protein-coding genes as well as noncoding regulatory sequences in animal genomes.
