Comprehensive optimization of a reporter assay toolbox for three distinct CRISPR‐Cas systems

Abstract

The CRISPR-associated protein system (CRISPR-Cas system) allows programmable gene editing through inducing double-strand breaks. Reporter assays for DNA cleavage and DNA repair events play an important role in advancing the CRISPR technology and improving our understanding of the underlying molecular mechanisms. Here, we developed a series of reporter assays to probe mechanisms of action of various editing processes, including non-homologous DNA end joining (NHEJ), homology-directed repair (HDR), and single-strand annealing (SSA).With special target design, the reporter assays as an optimized toolbox can be used to take advantage of three distinct CRISPR-Cas systems (SpCas9, SaCas9, and FnCpf1) and two different reporters (GFP and Gaussia luciferase). We further validated the Gaussia reporter assays using a series of small molecules, including NU7441, RI-1, and Mirin, and showcased the use of a GFP reporter assay as an effective tool for enrichment of cells with edited genome.