title: Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development.
abstract: Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic β-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.
authors.affiliation: Developmental Biology Program, Sloan Kettering Institute.
authors.forename: Zhong-Dong
authors.initials: ZD
authors.lastname: Shi
authors.affiliation: Developmental Biology Program, Sloan Kettering Institute.
authors.forename: Chew-Li
authors.initials: CL
authors.lastname: Soh
authors.affiliation: Developmental Biology Program, Sloan Kettering Institute; zhuzengrong@gmail.com.
authors.forename: Zengrong
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: Developmental Biology Program, Sloan Kettering Institute; huangfud@mskcc.org.
authors.forename: Danwei
authors.initials: D
authors.lastname: Huangfu
mesh_terms: D002454:Cell Differentiation; D059766:DNA End-Joining Repair; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D006801:Humans; D010179:Pancreas / Q000254:growth & development* / Q000378:metabolism; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000166:cytology*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems