Identifying critical transcriptional targets of the MYC oncogene using a novel competitive precision genome editing (CGE) assay

Abstract

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.

Lineage-specific oncogenes drive growth of major forms of human cancer using common downstream mechanisms

“…Consistently with previous publication 24 , the effect of mutating Y15 phosphorylation site in the CDK1 gene on fitness of HAP1 cells. For panels E and F, log2 values for day 8/day 2 ratios are shown for each sequence tag pair after calculating the ratio of read counts for mutated vs. original sequence at both timepoints.…”

“…Half of the cells were collected for gDNA isolation on day 2 after transfection, and the other half cultured until day 8 for late timepoint sample. Isolation of gDNA, treatment with RNase A, exonucleases I and VII, two-step PCR amplification, sequencing, and data analysis were performed as described in Pihlajamaa et al 24 . Read count 5-50 was used as a cutoff for day 2 samples depending on the sequencing depth.…”

“…We therefore tested whether phosphorylation of HK2 could explain the observed effects. We measured the requirement of known HK2 phosphorylation sites using competitive precision genome editing (CGE) assay (Fig 4D , E) 24 . We did not detect an effect on cell proliferation by mutation of 9 HK2 residue T473, whose phosphorylation has been reported to affect HK2 activity 25,26 .…”

“…CGE assay was performed as described in Pihlajamaa et al 24 . Briefly, 200,000-400,000 early-passage HAP1 cells were transfected with a RNP complex containing tracrRNA and crRNA (250-500ng) with S.p.…”