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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.
Highlights
Designer nucleases such as clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas[9] are efficient genome-editing tools that hold great promise for experimental biology and therapies[1,2,3]
Methods to detect homology-directed repair (HDR) and nonhomologous endjoining (NHEJ) rely on gel-based systems or artificial reporter assays—neither of which are suitable for systematic analysis of many editing conditions at endogenous gene loci[14,15,16,17]
We found that the frequency of HDR is very low compared to NHEJ in any conditions we tested in HeLa cells, demonstrating a clear cell type dependency of genome-editing outcomes (Fig. 4a,b, and Supplementary Fig. S9)
Summary
Designer nucleases such as clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas[9] are efficient genome-editing tools that hold great promise for experimental biology and therapies[1,2,3] These tools induce a nick or a double-strand break (DSB) at targeted regions to activate two DNA repair pathways: homology-directed repair (HDR) and nonhomologous end-joining (NHEJ). The targeting specificity of CRISPR-based systems was improved by the development of dual Cas[9] D10A nickase (Cas9-D10A) and paired catalytically dead Cas[9] fused to FokI (FokI-dCas9) systems[4,5,6,7] Those different nuclease platforms, including another type of Cas[9] nickases, Cas[9] H840A nickase (Cas9-H840A), have different modes of DNA nicking or cleavage. Owing to the limitations of detection methods, the activity of sequence-specific nucleases has been assessed mainly by detecting NHEJ4–7
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