Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing

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.

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

“…A high-throughput screen designed to identify potential small molecules capable of enhancing HDR-mediated genome editing has been used to discover additional compounds that can be administered to cells to increase precision genome editing (Yu et al, 2015). HDR is known to be dependent on many variables, such as the cell- and tissue-type, the location of the target DNA within the chromosome, and the cell cycle (Biswas et al, 1992; Saleh-Gohari and Helleday, 2004; Heyer et al, 2010; Miyaoka et al, 2016). Chemically synchronizing cells to arrest them in G-phase by blocking M-phase before delivering Cas9 RNP and donor DNA template will increase HDR rates (Lin et al, 2014b).…”

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

“…21, 48 We do not advocate the use of Cas9n for generating cardiovascular mouse models because of the inherent complexity in experimental design as well as the lower efficiency of SpyCas9n versus wild type SpyCas9 in mediating genome edits, 49 though editing genomes is highly locus-dependent and there may be instances where Cas9n is desirable. 50 On the other hand, newer generations of SpyCas9 carrying mutations that do not alter the two nuclease domains have been developed that also reduce off-targeting events. These include “enhanced specificity” SpyCas9 (https://www.addgene.org/71814/) 51 and a “high fidelity” SpyCas9 (https://www.addgene.org/72247/).…”

“…Recently, a digital PCR-based assay was reported for high resolution determination of specific edits in various cell culture systems. 50 In some cases, such as the integration of donor templates with long homology arms, Southern blotting may be needed to ensure proper genome editing. Below, we describe simple PCR-based assays, including one that often identifies single base pair edits introduced with 3c CRISPR.…”

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

“…A high-throughput screen designed to identify potential small molecules capable of enhancing HDR-mediated genome editing has been used to discover additional compounds that can be administered to cells to increase precision genome editing . HDR is known to be dependent on many variables, such as the cell-and tissue-type, the location of the target DNA within the chromosome, and the cell cycle (Biswas et al, 1992;Saleh-Gohari and Helleday, 2004;Heyer et al, 2010;Miyaoka et al, 2016). Chemically synchronizing cells to arrest them in G-phase by blocking M-phase before delivering Cas9 RNP and donor DNA template will increase HDR rates (Lin et al, 2014b).…”