Quantifying on- and off-target genome editing

Abstract

Genome editing with engineered nucleases is a rapidly growing field thanks to transformative technologies that allow researchers to precisely alter genomes for numerous applications including basic research, biotechnology, and human gene therapy. While the ability to make precise and controlled changes at specified sites throughout the genome has grown tremendously in recent years, we still lack a comprehensive and standardized battery of assays for measuring the different genome editing outcomes created at endogenous genomic loci. Here we review the existing assays for quantifying on- and off-target genome editing and describe their utility in advancing the technology. We also highlight unmet assay needs for quantifying on- and off-target genome editing outcomes and discuss their importance for the genome editing field.

Keywords: CRISPR/Cas9; RNA-guided endonucleases; TALENs; ZFNs; gene editing; gene targeting; homologous recombination; nonhomologous end-joining.

Figure 1. Assays for quantifying on-target genome editing outcomes

(A) Schematic of mutation detection assay using CEL-I/T7E1 enzyme to measure NHEJ. (B) Schematic of RFLP assay to measure HR. (C) Schematic of traffic light reporter assay to measure both NHEJ and HR. (D) Schematic of SMRT sequencing to measure both NHEJ and HR at endogenous loci. For more details, refer to the main text.

Figure 2. Locating nuclease off-target activity

Off-target sites are predicted through either in vitro, cellular, or in silico techniques, which generate a list of potential off-target sites. Genomic DNA is harvested from cells treated with nucleases, the sites are amplified, tested and a subset validated as bona fide off-target sites.