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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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE140911 | GEO | 2020/09/28
REPOSITORIES: GEO
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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.
| S-EPMC7681025 | biostudies-literature
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).
2020-09-28 | GSE140910 | GEO
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).
2020-09-28 | GSE140909 | GEO
Project description:Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.
| S-EPMC5374001 | biostudies-literature
Project description:A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells
| PRJNA591388 | ENA
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.
2016-05-01 | E-MTAB-4143 | biostudies-arrayexpress
Project description:A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells (sgRNA-seq)
| PRJNA591392 | ENA
Project description:A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans- regulatory elements of Oct4 in embryonic stem cells (ATAC-seq)
| PRJNA591393 | ENA
Project description:An enormous amount of tumor sequencing data has been generated through large scale sequencing efforts. The functional consequences of the majority of mutations identified by such projects remain an open, unexplored question. This problem is particularly complicated in the case of rare mutations where frequency of occurrence alone or prediction of functional consequences are insufficient to distinguish driver from passenger or bystander mutations. We combine genome editing technology with a powerful mouse cancer model to uncover previously unsuspected rare oncogenic mutations in Burkitt's lymphoma. We identify two candidate tumor suppressors whose loss cooperate with MYC over-expression to accelerate lymphomagenesis. Our results highlight the utility of in vivo CRISPR/Cas9 screens combined with powerful mouse models to identify and validate rare oncogenic modifier events from tumor mutational data.
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Project description:Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.
| S-EPMC4644101 | biostudies-literature