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

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus

PROVIDER: GSE64059 | GEO | 2015/03/03

SECONDARY ACCESSION(S): PRJNA269978

REPOSITORIES: GEO

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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 (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

2015-03-03 | E-GEOD-64057 | 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 (ATAC-Seq)

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 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. V6.5 mouse ES cells according to the protocol as described

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 (ATAC-Seq)

2015-03-03 | GSE64058 | GEO

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

| PRJNA269978 | ENA

Functional annotation of native enhancers with a Cas9-histone demethylase fusion.

Project description:Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type-specific function. Here, we use a nuclease-deficient Cas9 (dCas9)-histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.

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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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Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.

Project description:The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavage can be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA.

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Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States.

Project description:High-resolution Cas9 structures have yet to reveal catalytic conformations due to HNH nuclease domain positioning away from the cleavage site. Nme1Cas9 and Nme2Cas9 are compact nucleases for in vivo genome editing. Here, we report structures of meningococcal Cas9 homologs in complex with sgRNA, dsDNA, or the AcrIIC3 anti-CRISPR protein. DNA-bound structures represent an early step of target recognition, a later HNH pre-catalytic state, the HNH catalytic state, and a cleaved-target-DNA-bound state. In the HNH catalytic state of Nme1Cas9, the active site is seen poised at the scissile phosphodiester linkage of the target strand, providing a high-resolution view of the active conformation. The HNH active conformation activates the RuvC domain. Our structures explain how Nme1Cas9 and Nme2Cas9 read distinct PAM sequences and how AcrIIC3 inhibits Nme1Cas9 activity. These structures provide insights into Cas9 domain rearrangements, guide-target engagement, cleavage mechanism, and anti-CRISPR inhibition, facilitating the optimization of these genome-editing platforms.

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