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Cas9 Functionally Opens Chromatin.

Barkal AA, Srinivasan S, Hashimoto T, Gifford DK, Sherwood RI - PLoS ONE (2016)

Bottom Line: Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci.Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs.Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding.

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dCas9 induces chromatin accessibility at previously inaccessible genomic loci.a. mESCs were co-transfected with a Tol2 transposase (TPase), a Tol2 transposon-flanked dCas9 expression cassette, and a Tol2 transposon-flanked sgRNA cassette to yield stable expression of dCas9 and a sgRNA targeted to a region with inaccessible chromatin. b. 16/16 loci in previously inaccessible chromatin had statistically significant increases in DNase hypersensitivity (y-axis) upon sgRNA targeting as measured by DNase-qPCR (gray dots). DNase hypersensitivity at each locus is normalized to its level in the absence of sgRNA (blue dot), and the average normalized DNase hypersensitivity in the presence of gRNA for all loci is shown (red dot), which is statistically significantly increased over–sgRNA control. At least two replicates were performed for all conditions, and a two-tailed Student’s t-test used to calculate significance. c. DNase-qPCR measurement of DNase hypersensitivity (y-axis) is shown +/-150 bp from the sgRNA site (x-axis) at four targeted loci. DNase-qPCR values at each datapoint are normalized to hypersensitivity in the absence of sgRNA, and all loci are oriented such that the 20 bp sgRNA sequence is immediately to the left of 0 and the NGG PAM sequence is immediately to the right of 0. Three replicates were performed for all experiments.
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pone.0152683.g001: dCas9 induces chromatin accessibility at previously inaccessible genomic loci.a. mESCs were co-transfected with a Tol2 transposase (TPase), a Tol2 transposon-flanked dCas9 expression cassette, and a Tol2 transposon-flanked sgRNA cassette to yield stable expression of dCas9 and a sgRNA targeted to a region with inaccessible chromatin. b. 16/16 loci in previously inaccessible chromatin had statistically significant increases in DNase hypersensitivity (y-axis) upon sgRNA targeting as measured by DNase-qPCR (gray dots). DNase hypersensitivity at each locus is normalized to its level in the absence of sgRNA (blue dot), and the average normalized DNase hypersensitivity in the presence of gRNA for all loci is shown (red dot), which is statistically significantly increased over–sgRNA control. At least two replicates were performed for all conditions, and a two-tailed Student’s t-test used to calculate significance. c. DNase-qPCR measurement of DNase hypersensitivity (y-axis) is shown +/-150 bp from the sgRNA site (x-axis) at four targeted loci. DNase-qPCR values at each datapoint are normalized to hypersensitivity in the absence of sgRNA, and all loci are oriented such that the 20 bp sgRNA sequence is immediately to the left of 0 and the NGG PAM sequence is immediately to the right of 0. Three replicates were performed for all experiments.

Mentions: To address whether dCas9 is capable of opening previously closed chromatin, we built two Tol2 transposon-based vectors [15, 17] allowing stable expression of dCas9 and sgRNAs through transposon integration in mouse embryonic stem cells (mESC) (Fig 1A). dCas9 was fused with a V5 epitope tag, allowing us to confirm persistent, strong, nuclear expression (S1 Fig), and sgRNAs were modified to increase stability [13].


Cas9 Functionally Opens Chromatin.

Barkal AA, Srinivasan S, Hashimoto T, Gifford DK, Sherwood RI - PLoS ONE (2016)

dCas9 induces chromatin accessibility at previously inaccessible genomic loci.a. mESCs were co-transfected with a Tol2 transposase (TPase), a Tol2 transposon-flanked dCas9 expression cassette, and a Tol2 transposon-flanked sgRNA cassette to yield stable expression of dCas9 and a sgRNA targeted to a region with inaccessible chromatin. b. 16/16 loci in previously inaccessible chromatin had statistically significant increases in DNase hypersensitivity (y-axis) upon sgRNA targeting as measured by DNase-qPCR (gray dots). DNase hypersensitivity at each locus is normalized to its level in the absence of sgRNA (blue dot), and the average normalized DNase hypersensitivity in the presence of gRNA for all loci is shown (red dot), which is statistically significantly increased over–sgRNA control. At least two replicates were performed for all conditions, and a two-tailed Student’s t-test used to calculate significance. c. DNase-qPCR measurement of DNase hypersensitivity (y-axis) is shown +/-150 bp from the sgRNA site (x-axis) at four targeted loci. DNase-qPCR values at each datapoint are normalized to hypersensitivity in the absence of sgRNA, and all loci are oriented such that the 20 bp sgRNA sequence is immediately to the left of 0 and the NGG PAM sequence is immediately to the right of 0. Three replicates were performed for all experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816323&req=5

pone.0152683.g001: dCas9 induces chromatin accessibility at previously inaccessible genomic loci.a. mESCs were co-transfected with a Tol2 transposase (TPase), a Tol2 transposon-flanked dCas9 expression cassette, and a Tol2 transposon-flanked sgRNA cassette to yield stable expression of dCas9 and a sgRNA targeted to a region with inaccessible chromatin. b. 16/16 loci in previously inaccessible chromatin had statistically significant increases in DNase hypersensitivity (y-axis) upon sgRNA targeting as measured by DNase-qPCR (gray dots). DNase hypersensitivity at each locus is normalized to its level in the absence of sgRNA (blue dot), and the average normalized DNase hypersensitivity in the presence of gRNA for all loci is shown (red dot), which is statistically significantly increased over–sgRNA control. At least two replicates were performed for all conditions, and a two-tailed Student’s t-test used to calculate significance. c. DNase-qPCR measurement of DNase hypersensitivity (y-axis) is shown +/-150 bp from the sgRNA site (x-axis) at four targeted loci. DNase-qPCR values at each datapoint are normalized to hypersensitivity in the absence of sgRNA, and all loci are oriented such that the 20 bp sgRNA sequence is immediately to the left of 0 and the NGG PAM sequence is immediately to the right of 0. Three replicates were performed for all experiments.
Mentions: To address whether dCas9 is capable of opening previously closed chromatin, we built two Tol2 transposon-based vectors [15, 17] allowing stable expression of dCas9 and sgRNAs through transposon integration in mouse embryonic stem cells (mESC) (Fig 1A). dCas9 was fused with a V5 epitope tag, allowing us to confirm persistent, strong, nuclear expression (S1 Fig), and sgRNAs were modified to increase stability [13].

Bottom Line: Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci.Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs.Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding.

Show MeSH
Related in: MedlinePlus