title: Efficient generation of hiPSC neural lineage specific knockin reporters using the CRISPR/Cas9 and Cas9 double nickase system.
abstract: Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.
authors.affiliation: Department of Neurosurgery, The University of Texas Health Science Center at Houston; Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston.
authors.forename: Shenglan
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Neurosurgery, The University of Texas Health Science Center at Houston; Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston.
authors.forename: Haipeng
authors.initials: H
authors.lastname: Xue
authors.affiliation: Department of Neurosurgery, The University of Texas Health Science Center at Houston; Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston; Department of Anesthesiology, Shengjing Hospital, China Medical University.
authors.forename: Bo
authors.initials: B
authors.lastname: Long
authors.affiliation: Department of Neurosurgery, The University of Texas Health Science Center at Houston; Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston; Department of Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine.
authors.forename: Li
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Neurosurgery, The University of Texas Health Science Center at Houston; Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston; Summer Research Program, Office of Educational Programs, The University of Texas Health Science Center at Houston; Biology Department, University of West Georgia.
authors.forename: Tai
authors.initials: T
authors.lastname: Truong
authors.affiliation: Department of Neurosurgery, The University of Texas Health Science Center at Houston; Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston; The Senator Lloyd & B. A. Bentsen Center for Stroke Research, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston; Ying.Liu@uth.tmc.edu.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
mesh_terms: D019070:Cell Lineage; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000378:metabolism; D055879:Gene Knock-In Techniques / Q000379:methods*; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors; D059765:Homologous Recombination; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D058953:Neural Stem Cells / Q000166:cytology / Q000502:physiology*
chemical_list: D003850:Deoxyribonuclease I