Journal
STEM CELL REPORTS
Volume 4, Issue 6, Pages 1103-1111Publisher
CELL PRESS
DOI: 10.1016/j.stemcr.2015.04.016
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Funding
- NIH [R01DK096239]
- NYSTEM [C029156]
- Basil O'Connor Starter Scholar Award from March of Dimes Birth Defects Foundation
- Tri-Institutional Stem Cell Initiative
- Louis V. Gerstner Jr. Young Investigators Award
- MSKCC Society Special Projects Committee
- New York State Stem Cell Science (NYSTEM) fellowship from the Center for Stem Cell Biology of the Sloan Kettering Institute
- Howard Hughes Medical Institute (HHMI) Medical Research Fellowship
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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.
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