Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase

Sci Rep. 2016 Dec 2:6:38198. doi: 10.1038/srep38198.


Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • CRISPR-Cas Systems*
  • Gene Silencing*
  • Humans
  • Induced Pluripotent Stem Cells / metabolism*
  • Transcription, Genetic*