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Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation.

McDonald JI, Celik H, Rois LE, Fishberger G, Fowler T, Rees R, Kramer A, Martens A, Edwards JR, Challen GA - Biol Open (2016)

Bottom Line: DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs.We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene.These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.

View Article: PubMed Central - PubMed

Affiliation: Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, USA.

No MeSH data available.


Limited off-target activity in dCas9-D3A driven targeted DNA methylation system. (A) Global 5-mC content levels in cells transduced with only dCas9-D3A, dCas9-D3A with sgRNAs, or dCas9-mD3A with sgRNAs targeting Cdkn1a locus (n=2 biological replicates performed in technical duplicate; unpaired t-test). (B) Bisulfite sequencing analysis of off-target CpG islands located upstream or downstream of Cdkn1a target locus (Srsf3 and off-target Cdkn1a, respectively) or distant loci (Cdkn1b and Cdkn2d) in cells that are transduced with dCas9-3a or dCas9-3a with both sgRNAs (n=10-24). Source data in Table S5. Error bars=mean±s.e.m.
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BIO019067F6: Limited off-target activity in dCas9-D3A driven targeted DNA methylation system. (A) Global 5-mC content levels in cells transduced with only dCas9-D3A, dCas9-D3A with sgRNAs, or dCas9-mD3A with sgRNAs targeting Cdkn1a locus (n=2 biological replicates performed in technical duplicate; unpaired t-test). (B) Bisulfite sequencing analysis of off-target CpG islands located upstream or downstream of Cdkn1a target locus (Srsf3 and off-target Cdkn1a, respectively) or distant loci (Cdkn1b and Cdkn2d) in cells that are transduced with dCas9-3a or dCas9-3a with both sgRNAs (n=10-24). Source data in Table S5. Error bars=mean±s.e.m.

Mentions: To assess non-specific activity in this system, we initially attempted to analyze off-target genomic areas with high homology to predicted binding regions of both Cdkn1a sgRNAs. However, no regions with high homology were found as these sgRNAs were designed based on their minimal off-target binding and the presence of a PAM-site. As an alternative, we measured the global DNA methylation levels between cells that contained fusion proteins (dCas9-D3A or dCas9-mD3A) in the presence of both sgRNAs (Fig. 6A), as well as the local DNA methylation profile of CpG islands located upstream and downstream of the Cdkn1a target locus (Srsf3 and off-target Cdkn1a respectively), and the promoter CpG islands of the related cell cycle family members Cdkn1b and Cdkn2d (Fig. S6). Srsf3 and off-target Cdkn1a are adjacent to Cdkn1a target locus on chromosome 17 (chr17), whereas Cdkn1b (chr6) and Cdkn2d (chr4) are distant loci. We found no statistically significant difference in global 5-methylcytosine (5-mC) levels between different groups or across the regions that were analyzed (Fig. 6A,B).Fig. 6.


Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation.

McDonald JI, Celik H, Rois LE, Fishberger G, Fowler T, Rees R, Kramer A, Martens A, Edwards JR, Challen GA - Biol Open (2016)

Limited off-target activity in dCas9-D3A driven targeted DNA methylation system. (A) Global 5-mC content levels in cells transduced with only dCas9-D3A, dCas9-D3A with sgRNAs, or dCas9-mD3A with sgRNAs targeting Cdkn1a locus (n=2 biological replicates performed in technical duplicate; unpaired t-test). (B) Bisulfite sequencing analysis of off-target CpG islands located upstream or downstream of Cdkn1a target locus (Srsf3 and off-target Cdkn1a, respectively) or distant loci (Cdkn1b and Cdkn2d) in cells that are transduced with dCas9-3a or dCas9-3a with both sgRNAs (n=10-24). Source data in Table S5. Error bars=mean±s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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BIO019067F6: Limited off-target activity in dCas9-D3A driven targeted DNA methylation system. (A) Global 5-mC content levels in cells transduced with only dCas9-D3A, dCas9-D3A with sgRNAs, or dCas9-mD3A with sgRNAs targeting Cdkn1a locus (n=2 biological replicates performed in technical duplicate; unpaired t-test). (B) Bisulfite sequencing analysis of off-target CpG islands located upstream or downstream of Cdkn1a target locus (Srsf3 and off-target Cdkn1a, respectively) or distant loci (Cdkn1b and Cdkn2d) in cells that are transduced with dCas9-3a or dCas9-3a with both sgRNAs (n=10-24). Source data in Table S5. Error bars=mean±s.e.m.
Mentions: To assess non-specific activity in this system, we initially attempted to analyze off-target genomic areas with high homology to predicted binding regions of both Cdkn1a sgRNAs. However, no regions with high homology were found as these sgRNAs were designed based on their minimal off-target binding and the presence of a PAM-site. As an alternative, we measured the global DNA methylation levels between cells that contained fusion proteins (dCas9-D3A or dCas9-mD3A) in the presence of both sgRNAs (Fig. 6A), as well as the local DNA methylation profile of CpG islands located upstream and downstream of the Cdkn1a target locus (Srsf3 and off-target Cdkn1a respectively), and the promoter CpG islands of the related cell cycle family members Cdkn1b and Cdkn2d (Fig. S6). Srsf3 and off-target Cdkn1a are adjacent to Cdkn1a target locus on chromosome 17 (chr17), whereas Cdkn1b (chr6) and Cdkn2d (chr4) are distant loci. We found no statistically significant difference in global 5-methylcytosine (5-mC) levels between different groups or across the regions that were analyzed (Fig. 6A,B).Fig. 6.

Bottom Line: DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs.We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene.These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.

View Article: PubMed Central - PubMed

Affiliation: Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, USA.

No MeSH data available.