title: Generating a Genome Editing Nuclease for Targeted Mutagenesis in Human Cells.
abstract: Gene targeting and editing is an essential tool for both basic research and clinical application such as gene therapy. Several endonucleases have been invented to fulfill these purposes, including zinc finger nucleases, TALEN, and CRISPR/Cas9. Although all of these systems can target DNA sequence with high efficiency, they also exert off-target effects and genotoxicity. The off-target effects might not hinder their usage in animal models because the correctly targeted cells can be selected for further studies. However, the off-target effects could cause mutations which may be damaging or cancerous to the patients. In this chapter, we describe a genome-editing nuclease method which relies on modifying specific amino acids on a monomeric endonuclease, I-SceI, to recognize a targeted sequence in the human genome. This nuclease is small in size and shows a much lower genotoxicity compared to other nucleases including CRISPR/Cas9.
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, China.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: He
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, China. kkee@tsinghua.edu.cn.
authors.forename: Kehkooi
authors.initials: K
authors.lastname: Kee
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D004720:Endonucleases / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods; D015894:Genome, Human / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis / Q000235:genetics*
chemical_list: D004720:Endonucleases
keywords: Gene editing; Gene targeting; Homologous directed recombination; I-SceI; β-thalassemia