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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.


Site-specific induction of DNA methylation using a CRISPR-Cas9 DNMT fusion. (A) dCas9-DNMT3A CD fusion constructs. The E756A mutation inactivates the DNMT3A-CD. NLS, nuclear localization signal; FLAG, FLAG tag domain. (B) UCSC genome browser view showing the locations of the three CDKN2A sgRNA (g1a, g7a and g33a). The three sgRNA were validated to ensure they targeted this locus (Fig. S1.2). cDMR indicates the region from the literature where methylation changes are associated with expression changes (Fig. S1.1). (C) Induced DNA methylation at the CDKN2A promoter three days post-transfection. Colors correspond to the red and blue ABS regions in B. Three CpGs were independently measured in both amplicons. sgRNA target sites are indicated above the graphs. Pool sgRNA indicates g1a, g7a, g33a were used simultaneously. Sanger sequencing validation is presented in Fig. S1.3, and non-CpG methylation data is presented in Fig. S1.4. (D) Time course of the percent methylation data for the CpG marked with an asterisk in C. Additional CpGs are shown in Fig. S1.5. (E) Methylation induced by a pair of sgRNA decreases with increasing intervening distance. Distance is calculated relative to the 3′ end of the g33a sgRNA. Diamonds indicate the location each CpG monitored for methylation; whose color corresponds to appropriate line in the graph. Additional data from individual and paired sgRNA is presented in Fig. S1.6. (F) CDKN2A expression for samples with induced methylation. Expression is normalized to day one for each respective sample. Error bars=mean±s.e.m. (n=1 performed in technical triplicate).
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BIO019067F1: Site-specific induction of DNA methylation using a CRISPR-Cas9 DNMT fusion. (A) dCas9-DNMT3A CD fusion constructs. The E756A mutation inactivates the DNMT3A-CD. NLS, nuclear localization signal; FLAG, FLAG tag domain. (B) UCSC genome browser view showing the locations of the three CDKN2A sgRNA (g1a, g7a and g33a). The three sgRNA were validated to ensure they targeted this locus (Fig. S1.2). cDMR indicates the region from the literature where methylation changes are associated with expression changes (Fig. S1.1). (C) Induced DNA methylation at the CDKN2A promoter three days post-transfection. Colors correspond to the red and blue ABS regions in B. Three CpGs were independently measured in both amplicons. sgRNA target sites are indicated above the graphs. Pool sgRNA indicates g1a, g7a, g33a were used simultaneously. Sanger sequencing validation is presented in Fig. S1.3, and non-CpG methylation data is presented in Fig. S1.4. (D) Time course of the percent methylation data for the CpG marked with an asterisk in C. Additional CpGs are shown in Fig. S1.5. (E) Methylation induced by a pair of sgRNA decreases with increasing intervening distance. Distance is calculated relative to the 3′ end of the g33a sgRNA. Diamonds indicate the location each CpG monitored for methylation; whose color corresponds to appropriate line in the graph. Additional data from individual and paired sgRNA is presented in Fig. S1.6. (F) CDKN2A expression for samples with induced methylation. Expression is normalized to day one for each respective sample. Error bars=mean±s.e.m. (n=1 performed in technical triplicate).

Mentions: To design a flexible system to target DNA methylation, we fused dCas9 to the catalytic domain of the de novo DNA methyltransferase DNMT3A (Fig. 1A). To test this system we targeted DNA methylation to the tumor suppressor gene CDKN2A (cyclin dependent kinase 2A), which inhibits progression through the cell cycle (Liggett and Sidransky, 1998). CDKN2A is one of the most frequently hypermethylated genes in The Cancer Genome Atlas (Ciriello et al., 2013), and numerous clinical studies show a negative correlation between CDKN2A methylation and expression in colorectal cancer (Shima et al., 2011). While it is generally assumed that CDKN2A methylation induces gene silencing, it has also been suggested that DNA methylation occurs after the loss of expression (Hinshelwood et al., 2009). From a literature search, we identified 17 publications that associate CDKN2A methylation with expression and/or cancer (Fig. S1.1). Overwhelmingly, these papers studied the differentially methylated region (cancer DMR, cDMR) on the 3′ end of the CpG island that overlapped the first exon of CDKN2A (Fig. 1B).Fig. 1.


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)

Site-specific induction of DNA methylation using a CRISPR-Cas9 DNMT fusion. (A) dCas9-DNMT3A CD fusion constructs. The E756A mutation inactivates the DNMT3A-CD. NLS, nuclear localization signal; FLAG, FLAG tag domain. (B) UCSC genome browser view showing the locations of the three CDKN2A sgRNA (g1a, g7a and g33a). The three sgRNA were validated to ensure they targeted this locus (Fig. S1.2). cDMR indicates the region from the literature where methylation changes are associated with expression changes (Fig. S1.1). (C) Induced DNA methylation at the CDKN2A promoter three days post-transfection. Colors correspond to the red and blue ABS regions in B. Three CpGs were independently measured in both amplicons. sgRNA target sites are indicated above the graphs. Pool sgRNA indicates g1a, g7a, g33a were used simultaneously. Sanger sequencing validation is presented in Fig. S1.3, and non-CpG methylation data is presented in Fig. S1.4. (D) Time course of the percent methylation data for the CpG marked with an asterisk in C. Additional CpGs are shown in Fig. S1.5. (E) Methylation induced by a pair of sgRNA decreases with increasing intervening distance. Distance is calculated relative to the 3′ end of the g33a sgRNA. Diamonds indicate the location each CpG monitored for methylation; whose color corresponds to appropriate line in the graph. Additional data from individual and paired sgRNA is presented in Fig. S1.6. (F) CDKN2A expression for samples with induced methylation. Expression is normalized to day one for each respective sample. Error bars=mean±s.e.m. (n=1 performed in technical triplicate).
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BIO019067F1: Site-specific induction of DNA methylation using a CRISPR-Cas9 DNMT fusion. (A) dCas9-DNMT3A CD fusion constructs. The E756A mutation inactivates the DNMT3A-CD. NLS, nuclear localization signal; FLAG, FLAG tag domain. (B) UCSC genome browser view showing the locations of the three CDKN2A sgRNA (g1a, g7a and g33a). The three sgRNA were validated to ensure they targeted this locus (Fig. S1.2). cDMR indicates the region from the literature where methylation changes are associated with expression changes (Fig. S1.1). (C) Induced DNA methylation at the CDKN2A promoter three days post-transfection. Colors correspond to the red and blue ABS regions in B. Three CpGs were independently measured in both amplicons. sgRNA target sites are indicated above the graphs. Pool sgRNA indicates g1a, g7a, g33a were used simultaneously. Sanger sequencing validation is presented in Fig. S1.3, and non-CpG methylation data is presented in Fig. S1.4. (D) Time course of the percent methylation data for the CpG marked with an asterisk in C. Additional CpGs are shown in Fig. S1.5. (E) Methylation induced by a pair of sgRNA decreases with increasing intervening distance. Distance is calculated relative to the 3′ end of the g33a sgRNA. Diamonds indicate the location each CpG monitored for methylation; whose color corresponds to appropriate line in the graph. Additional data from individual and paired sgRNA is presented in Fig. S1.6. (F) CDKN2A expression for samples with induced methylation. Expression is normalized to day one for each respective sample. Error bars=mean±s.e.m. (n=1 performed in technical triplicate).
Mentions: To design a flexible system to target DNA methylation, we fused dCas9 to the catalytic domain of the de novo DNA methyltransferase DNMT3A (Fig. 1A). To test this system we targeted DNA methylation to the tumor suppressor gene CDKN2A (cyclin dependent kinase 2A), which inhibits progression through the cell cycle (Liggett and Sidransky, 1998). CDKN2A is one of the most frequently hypermethylated genes in The Cancer Genome Atlas (Ciriello et al., 2013), and numerous clinical studies show a negative correlation between CDKN2A methylation and expression in colorectal cancer (Shima et al., 2011). While it is generally assumed that CDKN2A methylation induces gene silencing, it has also been suggested that DNA methylation occurs after the loss of expression (Hinshelwood et al., 2009). From a literature search, we identified 17 publications that associate CDKN2A methylation with expression and/or cancer (Fig. S1.1). Overwhelmingly, these papers studied the differentially methylated region (cancer DMR, cDMR) on the 3′ end of the CpG island that overlapped the first exon of CDKN2A (Fig. 1B).Fig. 1.

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.