Back to Search
Start Over
Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.
- Source :
-
Scientific reports [Sci Rep] 2017 Nov 13; Vol. 7 (1), pp. 15480. Date of Electronic Publication: 2017 Nov 13. - Publication Year :
- 2017
-
Abstract
- We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.
- Subjects :
- Alleles
Diphtheria Toxin administration & dosage
Gene Knockout Techniques methods
Genetic Vectors genetics
Histidine analogs & derivatives
Histidine biosynthesis
Humans
MCF-7 Cells
Minor Histocompatibility Antigens metabolism
Proteins metabolism
Puromycin administration & dosage
Transfection methods
Transgenes genetics
Tumor Suppressor Proteins metabolism
CRISPR-Cas Systems genetics
Gene Editing methods
Minor Histocompatibility Antigens genetics
Proteins genetics
Tumor Suppressor Proteins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 7
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 29133816
- Full Text :
- https://doi.org/10.1038/s41598-017-15206-x