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Rapid functional annotation of native enhancers in embryonic stem cells with a Neisseria meningitidis CRISPR-Cas9 system (ATAC-Seq)

ABSTRACT: Gene regulatory elements such as enhancers have profound effects on cellular function, health and disease. Our understanding of mammalian enhancer function is limited by the lack of technology that would allow for a rapid and thorough test of their cell type-specific function. Here we describe a novel Cas9-effector system that enables rapid testing and functional annotation of native enhancers in embryonic stem cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE64058 | GEO | 2015/03/03

SECONDARY ACCESSION(S): PRJNA269982

REPOSITORIES: GEO

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Rapid functional annotation of native enhancers in embryonic stem cells with a Neisseria meningitidis CRISPR-Cas9 system (BeadChip)

Project description:Gene regulatory elements such as enhancers have profound effects on cellular function, health, and disease. Our understanding of mammalian enhancer function is limited by the lack of a technology that would allow for a rapid and thorough test of their cell type-specific function. Here, we describe a novel Cas9-effector system that enables rapid testing and functional annotation of native enhancers in embryonic stem cells. Total RNA obtained from R26 dCas9-effector mESC lines after viral delivery of sgRNAs directed against proximal promoter or enhancer regions

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Rapid functional annotation of native enhancers in embryonic stem cells with a Neisseria meningitidis CRISPR-Cas9 system (ATAC-Seq)

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2015-03-03 | E-GEOD-64058 | biostudies-arrayexpress

Rapid functional annotation of native enhancers in embryonic stem cells with a Neisseria meningitidis CRISPR-Cas9 system (BeadChip)

2015-03-03 | GSE64057 | GEO

Rapid functional annotation of native enhancers in embryonic stem cells with a Neisseria meningitidis CRISPR-Cas9 system

Project description:This SuperSeries is composed of the SubSeries listed below.

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Rapid functional annotation of native enhancers in embryonic stem cells with a Neisseria meningitidis CRISPR-Cas9 system

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2015-03-03 | E-GEOD-64059 | biostudies-arrayexpress

Rapid functional annotation of native enhancers in embryonic stem cells with a Neisseria meningitidis CRISPR-Cas9 system

Project description:Rapid functional annotation of native enhancers in embryonic stem cells with a Neisseria meningitidis CRISPR-Cas9 system

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DNase H Activity of Neisseria meningitidis Cas9.

Project description:Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs (including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 cleaves single-stranded DNAs in a manner that is RNA guided but PAM and tracrRNA independent. Beyond the need for guide-target pairing, this "DNase H" activity has no apparent sequence requirements, and the cleavage sites are measured from the 5' end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA is not strictly required for NmeCas9 enzymatic activation, and expand the list of targeting activities of Cas9 endonucleases.

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Improved production process for native outer membrane vesicle vaccine against Neisseria meningitidis.

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The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells.

Project description:The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications.

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