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Utilizing a dual endogenous reporter system to identify functional regulators of aberrant stem cell and differentiation activity in colorectal cancer

ABSTRACT: Utilizing a dual endogenous reporter system to identify functional regulators of aberrant stem cell and differentiation activity in colorectal cancer

PROVIDER: PRJNA989580 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25098063.fastq.gz	Fastqsanger.gz
	SRR25098064.fastq.gz	Fastqsanger.gz
	SRR25098065.fastq.gz	Fastqsanger.gz
	SRR25098066.fastq.gz	Fastqsanger.gz
	SRR25098067.fastq.gz	Fastqsanger.gz

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Utilizing a dual endogenous reporter system to identify functional regulators of aberrant stem cell and differentiation activity in colorectal cancer

Project description:Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators that regulate these key cellular programs in CRC, we developed an endogenous reporter system by genome-editing human CRC cell lines with knock-in fluorescent reporters at the SOX9 and KRT20 locus to report aberrant stem cell-like activity and differentiation respectively in pooled CRISPR screening formats. We then constructed a dual reporter system that simultaneously monitors aberrant stem cell-like and differentiation activity in the same CRC cell line, improving our signal to noise discrimination. Using a focused-library CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs, we identified factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominated SMARCB1 of the SWI/SNF BAF complex as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency in CRC and required for human CRC cell line and patient-derived organoid growth in vivo. These studies highlight the utility of a biologically designed endogenous reporter system to uncover novel therapeutic targets for drug development.

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Utilizing a dual endogenous reporter system to identify functional regulators of aberrant stem cell and differentiation activity in colorectal cancer.

Project description:Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators that regulate these key cellular programs in CRC, we developed an endogenous reporter system by genome-editing human CRC cell lines with knock-in fluorescent reporters at the SOX9 and KRT20 locus to report aberrant stem cell-like activity and differentiation, respectively, and then performed pooled genetic perturbation screens. Constructing a dual reporter system that simultaneously monitored aberrant stem cell-like and differentiation activity in the same CRC cell line improved our signal to noise discrimination. Using a focused-library CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs, we identified factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominated SMARCB1 of the BAF complex (also known as SWI/SNF) as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency in CRC and required for in vivo growth of human CRC models. These studies highlight the utility of a biologically designed endogenous reporter system to uncover novel therapeutic targets for drug development.

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Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system.

Project description:Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators of these key cellular programs, we engineer a dual endogenous reporter system by genome-editing the SOX9 and KRT20 loci of human CRC cell lines to express fluorescent reporters, broadcasting aberrant stem cell-like and differentiation activity, respectively. By applying a CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs to this platform, we identify factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominate SMARCB1 of the BAF complex (also known as SWI/SNF) as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency and required for in vivo growth of human CRC models. These studies highlight the utility of biologically designed endogenous reporter platforms to uncover regulators with therapeutic potential.

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