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Competitive precision genome editing (CGE) assay for functional analysis at single base resolution

The two major limitations of applying CRISPR/Cas9-technology for analysis of the effect of genotype on phenotype are the difficulty of cutting DNA exactly at the intended site, and the decreased cell proliferation and other phenotypic effects caused by the DNA cuts themselves. Here we report a novel competitive genome editing assay that allows analysis of the functional consequence of precise mutations. The method is based on precision genome editing, where a target sequence close to a feature of interest is cut, and the DNA is then repaired using a template that either reconstitutes the original feature, or introduces an altered sequence. Introducing sequence labels to both types of repair templates generates a large number of replicate cultures, increasing statistical power. In addition, the labels identify edited cells, allowing direct comparison between cells that carry wild-type and mutant features. Here, we apply the assay for multiplexed analysis of the role of E-box sequences on MYC binding and cellular fitness.