| First Author | Shy BR | Year | 2016 |
| Journal | Nucleic Acids Res | Volume | 44 |
| Issue | 16 | Pages | 7997-8010 |
| PubMed ID | 27484482 | Mgi Jnum | J:250352 |
| Mgi Id | MGI:5922048 | Doi | 10.1093/nar/gkw685 |
| Citation | Shy BR, et al. (2016) Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells. Nucleic Acids Res 44(16):7997-8010 |
| abstractText | CRISPR/Cas9 nucleases have enabled powerful, new genome editing capabilities; however, the preponderance of non-homologous end joining (NHEJ) mediated repair events over homology directed repair (HDR) in most cell types limits the ability to engineer precise changes in mammalian genomes. Here, we increase the efficiency of isolating precise HDR-mediated events in mouse embryonic stem (ES) cells by more than 20-fold through the use of co-incidental insertion (COIN) of independent donor DNA sequences. Analysis of on:off-target frequencies at the Lef1 gene revealed that bi-allelic insertion of a PGK-Neo cassette occurred more frequently than expected. Using various selection cassettes targeting multiple loci, we show that the insertion of a selectable marker at one control site frequently coincided with an insertion at an unlinked, independently targeted site, suggesting enrichment of a sub-population of HDR-proficient cells. When individual cell events were tracked using flow cytometry and fluorescent protein markers, individual cells frequently performed either a homology-dependent insertion event or a homology-independent event, but rarely both types of insertions in a single cell. Thus, when HDR-dependent selection donors are used, COIN enriches for HDR-proficient cells among heterogeneous cell populations. When combined with a self-excising selection cassette, COIN provides highly efficient and scarless genome editing. |
