title: Genome Editing in Human Pluripotent Stem Cells.
abstract: Pluripotent stem cells (PSCs), defined by their capacity for self-renewal and differentiation into all cell types, are an integral tool for basic biological research and disease modeling. However, full use of PSCs for research and regenerative medicine requires the ability to precisely edit their DNA to correct disease-causing mutations and for functional analysis of genetic variations. Recent advances in DNA editing of human stem cells (including PSCs) have benefited from the use of designer nucleases capable of making double-strand breaks (DSBs) at specific sequences that stimulate endogenous DNA repair. The clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become the preferred designer nuclease for genome editing in human PSCs and other cell types. Here we describe the principles for designing a single guide RNA to uniquely target a gene of interest and describe strategies for disrupting, inserting, or replacing a specific DNA sequence in human PSCs. The improvements in efficiency and ease provided by these techniques allow individuals to precisely engineer PSCs in a way previously limited to large institutes and core facilities.
authors.affiliation: Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Predoctoral Training Program in Human Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Cory
authors.initials: C
authors.lastname: Smith
authors.affiliation: Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205;
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Ye
authors.affiliation: Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Predoctoral Training Program in Human Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Linzhao
authors.initials: L
authors.lastname: Cheng
mesh_terms: D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting; D006801:Humans; D039904:Pluripotent Stem Cells; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems