Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing
Open Access
- 31 March 2016
- journal article
- Published by Springer Science and Business Media LLC in Scientific Reports
- Vol. 6 (1), 23549
- https://doi.org/10.1038/srep23549
Abstract
Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR–based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator–like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.Keywords
This publication has 38 references indexed in Scilit:
- mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5Nucleic Acids Research, 2015
- Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectorsNature Biotechnology, 2015
- The Mechanism of Gene Targeting in Human Somatic CellsPLoS Genetics, 2014
- Isolation of single-base genome-edited human iPS cells without antibiotic selectionNature Methods, 2014
- Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing SpecificityCell, 2013
- CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineeringNature Biotechnology, 2013
- A robust method to derive functional neural crest cells from human pluripotent stem cells.2013
- Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targetingNucleic Acids Research, 2011
- A more efficient method to generate integration-free human iPS cellsNature Methods, 2011
- Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design toolNucleic Acids Research, 2007