title: A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells.
abstract: The development of new gene-editing tools, in particular the CRISPR/Cas system, has greatly facilitated site-specific mutagenesis in human embryonic stem cells (hESCs), including the introduction or correction of patient-specific mutations for disease modeling. However, integration of a reporter gene into an endogenous locus in hESCs still requires a lengthy and laborious two-step strategy that involves first drug selection to identify correctly targeted clones and then excision of the drug-resistance cassette. Through the use of iCRISPR, an efficient gene-editing platform we recently developed, this study demonstrates a knockin strategy without drug selection for both active and silent genes in hESCs. Lineage-specific hESC reporter lines are useful for real-time monitoring of cell-fate decisions and lineage tracing, as well as enrichment of specific cell populations during hESC differentiation. Thus, this selection-free knockin strategy is expected to greatly facilitate the use of hESCs for developmental studies, disease modeling, and cell-replacement therapy.
authors.affiliation: Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Zengrong
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA; Weill Graduate School of Medical Sciences at Cornell University/The Rockefeller University/Sloan Kettering Institute Tri-Institutional M.D.-Ph.D. Program, 1300 York Avenue, New York, NY 10065, USA.
authors.forename: Nipun
authors.initials: N
authors.lastname: Verma
authors.affiliation: Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Federico
authors.initials: F
authors.lastname: González
authors.affiliation: Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Zhong-Dong
authors.initials: ZD
authors.lastname: Shi
authors.affiliation: Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA. Electronic address: huangfud@mskcc.org.
authors.forename: Danwei
authors.initials: D
authors.lastname: Huangfu
mesh_terms: D000483:Alleles; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D016415:Sequence Alignment; D015534:Trans-Activators / Q000235:genetics
chemical_list: D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein