title: Efficient and allele-specific genome editing of disease loci in human iPSCs.
abstract: Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.
authors.affiliation: 1] Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [2] Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [3] Predoctoral Training Program in Human Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Cory
authors.initials: C
authors.lastname: Smith
authors.affiliation: 1] Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [2] Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [3] Cellular and Molecular Medicine Graduate Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Leire
authors.initials: L
authors.lastname: Abalde-Atristain
authors.affiliation: 1] Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [2] Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Chaoxia
authors.initials: C
authors.lastname: He
authors.affiliation: Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Brett R
authors.initials: BR
authors.lastname: Brodsky
authors.affiliation: Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Evan M
authors.initials: EM
authors.lastname: Braunstein
authors.affiliation: 1] Cellular and Molecular Medicine Graduate Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [2] Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Pooja
authors.initials: P
authors.lastname: Chaudhari
authors.affiliation: 1] Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [2] Cellular and Molecular Medicine Graduate Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [3] Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Yoon-Young
authors.initials: YY
authors.lastname: Jang
authors.affiliation: 1] Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [2] Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [3] Predoctoral Training Program in Human Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [4] Cellular and Molecular Medicine Graduate Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Linzhao
authors.initials: L
authors.lastname: Cheng
authors.affiliation: 1] Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [2] Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Ye
mesh_terms: D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D059766:DNA End-Joining Repair; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015894:Genome, Human; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000473:pathology; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D040641:Quantitative Trait Loci; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D059767:Recombinational DNA Repair; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D000515:alpha 1-Antitrypsin; D053614:Janus Kinase 2; D000076987:CRISPR-Associated Protein 9; C000600049:Cas9 protein, Francisella novicida; D004720:Endonucleases