A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells

Stem Cell Reports. 2015 Jun 9;4(6):1103-11. doi: 10.1016/j.stemcr.2015.04.016. Epub 2015 May 28.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Base Sequence
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Gene Knock-In Techniques
  • Genes, Reporter
  • Homeodomain Proteins / genetics
  • Human Embryonic Stem Cells / cytology
  • Human Embryonic Stem Cells / metabolism*
  • Humans
  • Octamer Transcription Factor-3 / genetics
  • Sequence Alignment
  • Trans-Activators / genetics


  • Homeodomain Proteins
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • Trans-Activators
  • pancreatic and duodenal homeobox 1 protein