title: Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells.
abstract: To correct a DNA mutation in the human genome for gene therapy, homology-directed repair (HDR) needs to be specific and have the lowest off-target effects to protect the human genome from deleterious mutations. Zinc finger nucleases, transcription activator-like effector nuclease (TALEN) and CRISPR-CAS9 systems have been engineered and used extensively to recognize and modify specific DNA sequences. Although TALEN and CRISPR/CAS9 could induce high levels of HDR in human cells, their genotoxicity was significantly higher. Here, we report the creation of a monomeric endonuclease that can recognize at least 33 bp by fusing the DNA-recognizing domain of TALEN (TALE) to a re-engineered homing endonuclease I-SceI. After sequentially re-engineering I-SceI to recognize 18 bp of the human β-globin sequence, the re-engineered I-SceI induced HDR in human cells. When the re-engineered I-SceI was fused to TALE (TALE-ISVB2), the chimeric endonuclease induced the same HDR rate at the human β-globin gene locus as that induced by TALEN, but significantly reduced genotoxicity. We further demonstrated that TALE-ISVB2 specifically targeted at the β-globin sequence in human hematopoietic stem cells. Therefore, this monomeric endonuclease has the potential to be used in therapeutic gene targeting in human cells.
authors.affiliation: College of Life Sciences, Peking University, Beijing 100871, China Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China Tsinghua-Peking-NIBS Center for Life Sciences, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Jianfei
authors.initials: J
authors.lastname: Lin
authors.affiliation: College of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: He
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Hematology, the First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Luo
authors.affiliation: Department of Hematology, the First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China.
authors.forename: Yongrong
authors.initials: Y
authors.lastname: Lai
authors.affiliation: THU-PKU Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xie
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China Tsinghua-Peking-NIBS Center for Life 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; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015894:Genome, Human; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D059767:Recombinational DNA Repair; D052416:Targeted Gene Repair; D055544:beta-Globins / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D055544:beta-Globins; D004706:Endodeoxyribonucleases