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A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells (ATAC-seq)

ABSTRACT: A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells (ATAC-seq)

PROVIDER: PRJNA591393 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10523624_1.fastq.gz	Fastqsanger.gz
	SRR10523624_2.fastq.gz	Fastqsanger.gz
	SRR10523625_1.fastq.gz	Fastqsanger.gz
	SRR10523625_2.fastq.gz	Fastqsanger.gz
	SRR10523626_1.fastq.gz	Fastqsanger.gz

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A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells (sgRNA-seq)

Project description:Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the non-coding genome and control gene expression programs either in –cis or in –trans. Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ~12kb cis-region of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. The ctSCAN-SMS identified 10 CREs and 12 TREs, as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletion of these REs confirmed that the majority of the CREs and TREs are functionally active, and involved in regulating Oct4 gene expression. Additionally, a subset of the functional CREs and TREs physically interact with the Oct4 promoter to varying degrees through intra- and inter-chromosomal interactions, respectively. Comparative genomics analysis reveals that functional CREs are more conserved in terms of their regulatory sequence conservation between mouse and primates (including humans) than TREs. Notably, a few active CREs are devoid of canonical regulatory features. Taken together, our work demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs, and probe their roles in the regulation of transcriptional output of a target gene (in this case Oct4).

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A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells (ATAC-seq)

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A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells

Project description:A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells

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Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.

Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.

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A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells (sgRNA-seq)

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A saturating mutagenesis CRISPR-Cas9-mediated functional genomic screen identifies cis- and trans-regulatory elements of Oct4 in murine ESCs.

Project description:Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in cis or in trans Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ?12-kb cis-region (cis-REs; CREs) of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling Oct4 gene expression. A subset of active CREs and TREs physically interact with the Oct4 promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the Oct4 promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case Oct4) in their native context.

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A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells

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