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A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites

ABSTRACT: A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites

PROVIDER: PRJNA849144 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19648006.fastq.gz	Fastqsanger.gz
	SRR19648007.fastq.gz	Fastqsanger.gz
	SRR19648008.fastq.gz	Fastqsanger.gz
	SRR19648009.fastq.gz	Fastqsanger.gz

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A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites

Project description:Many biological processes are controlled by master regulators that activate complex downstream transcriptional and/or posttranslational networks. These networks have been hard to dissect using reverse genetics because of the difficulty of mutating large numbers of binding motifs or phosphorylation sites at different chromosomal loci. Here, we use a novel competitive genome editing (CGE) assay for dissecting the transcriptional network downstream of the master regulatory oncogene MYC. The CGE method is based on precision genome editing using template libraries that either reconstitute the original feature or introduce an altered sequence. Both types of libraries also introduce sequence tags, generating a large number of replicate lineages and enabling direct comparison between cells that carry original and mutant features. By using precision editing and by comparing two populations of edited cells, the CGE method overcomes the two main limitations of CRISPR/Cas9-technology in analyzing the effect of genotype on phenotype: 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.

2022-07-11 | GSE206080 | GEO

A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites.

Project description:Here we describe a competitive genome editing method that measures the effect of mutations on molecular functions, based on precision CRISPR editing using template libraries with either the original or altered sequence, and a sequence tag, enabling direct comparison between original and mutated cells. Using the example of the MYC oncogene, we identify important transcriptional targets and show that E-box mutations at MYC target gene promoters reduce cellular fitness.

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