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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.


Related in: MedlinePlus

Multiple sgRNAs are required for the efficient establishment of targeted methylation. (A) Average DNA methylation level of target locus within Cdkn1a promoter (n=12-15 biological replicates representative of two independent experiments; unpaired t-test). Presence or absence of each sgRNA in each clone is shown (−; +) at the bottom of the graph. (B) Expression level of Cdkn1a mRNA in 32D cells transduced with sgRNAs, dCas9-D3A, dCas9-mD3A with sgRNAs or dCas9-D3A with either one or both sgRNAs (n=2 biological replicates performed in technical duplicate; unpaired t-test). Presence or absence of each sgRNA in each clone is shown (−; +). (C) Average promoter DNA methylation (n=12-15 biological replicates representative of two independent experiments) or (D) expression level of Cdkn1a in 32D cells in 32D cells with one sgRNA which were then transduced with their complementary sgRNA (n=2 biological replicates performed in technical duplicate; unpaired t-test). (E) Average DNA methylation of Cdkn1a target locus for clones following doxycycline withdrawal for eight days. Clones were previously induced for 12 days, with starting induced DNA methylation level shown by red error bars (n=12-16 biological replicates representative of two independent experiments). (F) Expression level of Cdkn1a in clones after eight days of de-induction (n=2 biological replicates performed in technical duplicate; unpaired t-test). *P<0.05; **P<0.01; ****P<0.0001, error bars=mean±s.e.m.
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BIO019067F5: Multiple sgRNAs are required for the efficient establishment of targeted methylation. (A) Average DNA methylation level of target locus within Cdkn1a promoter (n=12-15 biological replicates representative of two independent experiments; unpaired t-test). Presence or absence of each sgRNA in each clone is shown (−; +) at the bottom of the graph. (B) Expression level of Cdkn1a mRNA in 32D cells transduced with sgRNAs, dCas9-D3A, dCas9-mD3A with sgRNAs or dCas9-D3A with either one or both sgRNAs (n=2 biological replicates performed in technical duplicate; unpaired t-test). Presence or absence of each sgRNA in each clone is shown (−; +). (C) Average promoter DNA methylation (n=12-15 biological replicates representative of two independent experiments) or (D) expression level of Cdkn1a in 32D cells in 32D cells with one sgRNA which were then transduced with their complementary sgRNA (n=2 biological replicates performed in technical duplicate; unpaired t-test). (E) Average DNA methylation of Cdkn1a target locus for clones following doxycycline withdrawal for eight days. Clones were previously induced for 12 days, with starting induced DNA methylation level shown by red error bars (n=12-16 biological replicates representative of two independent experiments). (F) Expression level of Cdkn1a in clones after eight days of de-induction (n=2 biological replicates performed in technical duplicate; unpaired t-test). *P<0.05; **P<0.01; ****P<0.0001, error bars=mean±s.e.m.

Mentions: We reverse cloned viral integrants to identify clones that contained either one of the two guides, or both sgRNAs together with dCas9-D3A. No induced promoter DNA methylation or reduction in gene expression was observed in clones expressing only a single sgRNA (Fig. 5A,B). In contrast, Cdkn1a promoter methylation was increased and its expression decreased in clones that expressed both sgRNAs (Fig. 5A,B). To confirm the presence of both sgRNAs were required to execute induced DNA methylation, clones that contained only a single sgRNA were transduced with their complementary sgRNA. Following this, after eight-days of induction, Cdkn1a DNA methylation increased across the promoter by up to 33% compared to the parental clone that contained only a single sgRNA (Fig. 5C). Cdkn1a expression was reduced by 40-50% in the complemented clones (Fig. 5D). These data indicate that targeting with multiple sgRNAs results in a synergism and induces robust DNA methylation. We also examined the stability of the induced DNA methylation by withdrawing doxycycline for eight days. Cdkn1a promoter DNA methylation and expression profiles remained largely unchanged (Fig. 5E,F) even though expression of dCas9-D3A was silenced (Fig. S4.2).Fig. 5.


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)

Multiple sgRNAs are required for the efficient establishment of targeted methylation. (A) Average DNA methylation level of target locus within Cdkn1a promoter (n=12-15 biological replicates representative of two independent experiments; unpaired t-test). Presence or absence of each sgRNA in each clone is shown (−; +) at the bottom of the graph. (B) Expression level of Cdkn1a mRNA in 32D cells transduced with sgRNAs, dCas9-D3A, dCas9-mD3A with sgRNAs or dCas9-D3A with either one or both sgRNAs (n=2 biological replicates performed in technical duplicate; unpaired t-test). Presence or absence of each sgRNA in each clone is shown (−; +). (C) Average promoter DNA methylation (n=12-15 biological replicates representative of two independent experiments) or (D) expression level of Cdkn1a in 32D cells in 32D cells with one sgRNA which were then transduced with their complementary sgRNA (n=2 biological replicates performed in technical duplicate; unpaired t-test). (E) Average DNA methylation of Cdkn1a target locus for clones following doxycycline withdrawal for eight days. Clones were previously induced for 12 days, with starting induced DNA methylation level shown by red error bars (n=12-16 biological replicates representative of two independent experiments). (F) Expression level of Cdkn1a in clones after eight days of de-induction (n=2 biological replicates performed in technical duplicate; unpaired t-test). *P<0.05; **P<0.01; ****P<0.0001, error bars=mean±s.e.m.
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Related In: Results  -  Collection

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BIO019067F5: Multiple sgRNAs are required for the efficient establishment of targeted methylation. (A) Average DNA methylation level of target locus within Cdkn1a promoter (n=12-15 biological replicates representative of two independent experiments; unpaired t-test). Presence or absence of each sgRNA in each clone is shown (−; +) at the bottom of the graph. (B) Expression level of Cdkn1a mRNA in 32D cells transduced with sgRNAs, dCas9-D3A, dCas9-mD3A with sgRNAs or dCas9-D3A with either one or both sgRNAs (n=2 biological replicates performed in technical duplicate; unpaired t-test). Presence or absence of each sgRNA in each clone is shown (−; +). (C) Average promoter DNA methylation (n=12-15 biological replicates representative of two independent experiments) or (D) expression level of Cdkn1a in 32D cells in 32D cells with one sgRNA which were then transduced with their complementary sgRNA (n=2 biological replicates performed in technical duplicate; unpaired t-test). (E) Average DNA methylation of Cdkn1a target locus for clones following doxycycline withdrawal for eight days. Clones were previously induced for 12 days, with starting induced DNA methylation level shown by red error bars (n=12-16 biological replicates representative of two independent experiments). (F) Expression level of Cdkn1a in clones after eight days of de-induction (n=2 biological replicates performed in technical duplicate; unpaired t-test). *P<0.05; **P<0.01; ****P<0.0001, error bars=mean±s.e.m.
Mentions: We reverse cloned viral integrants to identify clones that contained either one of the two guides, or both sgRNAs together with dCas9-D3A. No induced promoter DNA methylation or reduction in gene expression was observed in clones expressing only a single sgRNA (Fig. 5A,B). In contrast, Cdkn1a promoter methylation was increased and its expression decreased in clones that expressed both sgRNAs (Fig. 5A,B). To confirm the presence of both sgRNAs were required to execute induced DNA methylation, clones that contained only a single sgRNA were transduced with their complementary sgRNA. Following this, after eight-days of induction, Cdkn1a DNA methylation increased across the promoter by up to 33% compared to the parental clone that contained only a single sgRNA (Fig. 5C). Cdkn1a expression was reduced by 40-50% in the complemented clones (Fig. 5D). These data indicate that targeting with multiple sgRNAs results in a synergism and induces robust DNA methylation. We also examined the stability of the induced DNA methylation by withdrawing doxycycline for eight days. Cdkn1a promoter DNA methylation and expression profiles remained largely unchanged (Fig. 5E,F) even though expression of dCas9-D3A was silenced (Fig. S4.2).Fig. 5.

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.


Related in: MedlinePlus