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bioRxiv · 10.1101/2021.09.17.460746

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

Pihlajamaa, P.·Kauko, O.·Sahu, B.·Kivioja, T.·Taipale, J.
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Abstract

MYC is an oncogenic transcription factor that controls major pathways promoting cell growth and proliferation. MYC has been implicated in the regulation of large number of genes, but the exact target genes responsible for its proliferative effects are still not known. Here, we use a novel competitive genome editing (CGE) assay for studying the functional consequence of precise mutations of MYC binding sites on cell proliferation. The CGE method is based on precision genome editing, where a CRISPR/Cas9-induced DNA break is repaired using a template that either reconstitutes the original feature or introduces an altered sequence. Both types of repair templates harbor sequence tags that allow direct comparison between cells that carry original and mutant features and generate a large number of replicate cultures. The CGE method overcomes the limitations of CRISPR/Cas9-technology in analyzing the effect of genotype on phenotype, namely the difficulty of cutting DNA exactly at the intended site, and the decreased cell proliferation caused by the DNA cuts themselves. Importantly, it provides a powerful method for studying subtle effects elicited by mutation of individual transcription factor binding sites. We show here that E-box mutations at several MYC target gene promoters resulted in reduced cellular fitness, demonstrating a direct correlation between MYC-regulated cellular processes and MYC binding and identifying important transcriptional targets responsible for its functions.

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Pihlajamaa, P., Kauko, O., Sahu, B., Kivioja, T., Taipale, J.. 2021-09-17. Competitive precision genome editing (CGE) assay for functional analysis at single base resolution. https://doi.org/10.1101/2021.09.17.460746

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