Dataset Information

Site-specific recruitment of epigenetic factors with a modular CRISPR/Cas system.

ABSTRACT: Dissecting the complex network of epigenetic modifications requires tools that combine precise recognition of DNA sequences with the capability to modify epigenetic marks. The CRISPR/Cas system has been proven to be a valuable addition to existing methodologies that fulfill these tasks. So far, sequence-specific editing of epigenetic modifications such as DNA methylation and histone posttranslational modifications relied on direct fusions of enzymatically inactivated Cas9 (dCas9) with epigenetic effectors. Here, we report a novel, modular system that facilitates the recruitment of any GFP-tagged protein to desired genomic loci. By fusing dCas9 to a GFP-binding nanobody (GBP) we demonstrate that prevalent epigenetic modifications at mouse major satellite repeats can be erased or set de novo by recruiting GFP-coupled catalytic domains of TET1 and DNMT3A, respectively. Furthermore, we construct an inducible expression system that enables a temporally controlled expression of both GBP-dCas9 and the effector protein. Thus, our approach further expands the CRISPR/Cas toolbox for site-specific manipulation of epigenetic modifications with a modular and easy-to-use system.

SUBMITTER: Anton T

PROVIDER: S-EPMC5499900 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Site-specific recruitment of epigenetic factors with a modular CRISPR/Cas system.

Anton Tobias T Bultmann Sebastian S

Nucleus (Austin, Tex.) 20170223 3

Dissecting the complex network of epigenetic modifications requires tools that combine precise recognition of DNA sequences with the capability to modify epigenetic marks. The CRISPR/Cas system has been proven to be a valuable addition to existing methodologies that fulfill these tasks. So far, sequence-specific editing of epigenetic modifications such as DNA methylation and histone posttranslational modifications relied on direct fusions of enzymatically inactivated Cas9 (dCas9) with epigenetic ...[more]

PMID: 28448738

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Project description:BACKGROUND:The presence and activity of CRISPR-Cas defense systems is a hallmark of many prokaryotic microorganisms. Here, the distribution of sequences related to the highly iterated palindrome 1 (HIP1) element and the DNA methylation of CGATCG motifs embedded within HIP1 as a vital part of the CRISPR1 repeat sequence was analyzed in the cyanobacterium Synechocystis sp. PCC 6803. Previously suggested functions of HIP1 include organization of chromosomal structure, DNA recombination or gene regulation, all of which could be relevant in CRISPR-Cas functionality. RESULTS:The CRISPR1 repeat-spacer array contains more than 50 CGATCG elements that are double-methylated (5mCG6mATCG) by the enzymes M.Ssp6803I and M.Ssp6803III. Hence, more than 200 possible methylation events cluster over a stretch of 3600 bp of double-stranded DNA. Bisulfite sequencing showed that these motifs were highly methylated at the m5CGATCG positions whereas specific motifs within the CRISPR1 cas genes were hypomethylated suggesting a lowered accessibility for the DNA methylase to these regions. Assays for conjugation and CRISPR1-mediated DNA interference revealed a 50% drop in conjugation efficiency in the mutant lacking the 5mC methylation of CGATCG motifs, while the highly efficient DNA interference activity was not affected by the lack of m5CGATCG DNA-methylation, nor was the capability to differentiate between self and non-self targets based on the protospacer adjacent motifs (PAMs) GTA and GTC versus the non-PAM AGC. A third DNA methylation mediated by M.Ssp6803II modifies the first cytosine in the motif GGCC yielding GGm4CC. We found a remarkable absence of GGCC motifs and hence the corresponding methylation over an 11 kb stretch encompassing all the cas genes involved in interference and crRNA maturation but not adaptation of the CRISPR1 system. CONCLUSIONS:The lack of GGCC tetranucleotides along the CRISPR1 interference and maturation genes supports the reported hybrid character of subtype I-D CRISPR-Cas systems. We report tight and very high 5mC methylation of the CRISPR1 repeat sequences. Nevertheless, cells lacking the 5mC methylation activity were unaffected in their CRISPR1-mediated interference response but the efficiency of conjugation was reduced by 50%. These results point to an unknown role of m5CGATCG DNA-methylation marks in conjugation and DNA transformation.

Dataset Information

Site-specific recruitment of epigenetic factors with a modular CRISPR/Cas system.

Publications

Site-specific recruitment of epigenetic factors with a modular CRISPR/Cas system.

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