A CRISPR-based Approach for Proteomic Analysis of a Single Genomic Locus

Overview

Journal Epigenetics

Specialty Genetics

Date 2014 Aug 23

PMID 25147920

Citations 39

Authors

Zachary J Waldrip

Stephanie D Byrum

Aaron J Storey

Jun Gao

Alicia K Byrd

Samuel G Mackintosh

Wayne P Wahls

Sean D Taverna

Kevin D Raney

Alan J Tackett

Affiliations

Soon will be listed here.

Abstract

Any given chromosomal activity (e.g., transcription) is governed predominantly by the local epiproteome. However, defining local epiproteomes has been limited by a lack of effective technologies to isolate discrete sections of chromatin and to identify with precision specific proteins and histone posttranslational modifications (PTMs). We report the use of the Cas9 and guide RNA (gRNA) components of the CRISPR system for gRNA-directed purification of a discrete section of chromatin. Quantitative mass spectrometry provides for unambiguous identification of proteins and histone PTMs specifically associated with the enriched chromatin. This CRISPR-based Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) approach revealed changes in the local epiproteome of a promoter during activation of transcription. CRISPR-ChAP-MS thus has broad applications for discovering molecular components and dynamic regulation of any in vivo activity at a given chromosomal location.

Citing Articles

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The chaperone protein p32 stabilizes HIV-1 Tat and strengthens the p-TEFb/RNAPII/TAR complex promoting HIV transcription elongation.

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Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE.

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References

Zybailov B, Mosley A, Sardiu M, Coleman M, Florens L, Washburn M . Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J Proteome Res. 2006; 5(9):2339-47. DOI: 10.1021/pr060161n. View

Hamperl S, Brown C, Garea A, Perez-Fernandez J, Bruckmann A, Huber K . Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae. Nucleic Acids Res. 2013; 42(1):e2. PMC: 3874202. DOI: 10.1093/nar/gkt891. View

Byrum S, Raman A, Taverna S, Tackett A . ChAP-MS: a method for identification of proteins and histone posttranslational modifications at a single genomic locus. Cell Rep. 2012; 2(1):198-205. PMC: 3408609. DOI: 10.1016/j.celrep.2012.06.019. View

Dejardin J, Kingston R . Purification of proteins associated with specific genomic Loci. Cell. 2009; 136(1):175-86. PMC: 3395431. DOI: 10.1016/j.cell.2008.11.045. View

Byrum S, Taverna S, Tackett A . Quantitative analysis of histone exchange for transcriptionally active chromatin. J Clin Bioinforma. 2011; 1(1):17. PMC: 3164610. DOI: 10.1186/2043-9113-1-17. View

Mali P, Esvelt K, Church G . Cas9 as a versatile tool for engineering biology. Nat Methods. 2013; 10(10):957-63. PMC: 4051438. DOI: 10.1038/nmeth.2649. View

Byrum S, Taverna S, Tackett A . Purification of a specific native genomic locus for proteomic analysis. Nucleic Acids Res. 2013; 41(20):e195. PMC: 3814360. DOI: 10.1093/nar/gkt822. View

Byrum S, Larson S, Avaritt N, Moreland L, Mackintosh S, Cheung W . Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma. J Proteomics Bioinform. 2013; 6(3):43-50. PMC: 3748992. DOI: 10.4172/jpb.1000260. View

Fu Y, Foden J, Khayter C, Maeder M, Reyon D, Joung J . High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013; 31(9):822-6. PMC: 3773023. DOI: 10.1038/nbt.2623. View

10.

DiCarlo J, Norville J, Mali P, Rios X, Aach J, Church G . Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 2013; 41(7):4336-43. PMC: 3627607. DOI: 10.1093/nar/gkt135. View

11.

Scholze H, Boch J . TAL effectors are remote controls for gene activation. Curr Opin Microbiol. 2011; 14(1):47-53. DOI: 10.1016/j.mib.2010.12.001. View

12.

Griesenbeck J, Boeger H, Strattan J, Kornberg R . Affinity purification of specific chromatin segments from chromosomal loci in yeast. Mol Cell Biol. 2003; 23(24):9275-82. PMC: 309680. DOI: 10.1128/MCB.23.24.9275-9282.2003. View

13.

Fujita T, Fujii H . Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem Biophys Res Commun. 2013; 439(1):132-6. DOI: 10.1016/j.bbrc.2013.08.013. View

14.

Unnikrishnan A, Gafken P, Tsukiyama T . Dynamic changes in histone acetylation regulate origins of DNA replication. Nat Struct Mol Biol. 2010; 17(4):430-7. PMC: 3060656. DOI: 10.1038/nsmb.1780. View

15.

Jiang W, Bikard D, Cox D, Zhang F, Marraffini L . RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31(3):233-9. PMC: 3748948. DOI: 10.1038/nbt.2508. View

16.

Hoshino A, Fujii H . Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. J Biosci Bioeng. 2009; 108(5):446-9. DOI: 10.1016/j.jbiosc.2009.05.005. View

17.

Akiyoshi B, Nelson C, Ranish J, Biggins S . Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit. Genes Dev. 2009; 23(24):2887-99. PMC: 2800092. DOI: 10.1101/gad.1865909. View

18.

Dai B, Rasmussen T . Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells. Stem Cells. 2007; 25(10):2567-74. DOI: 10.1634/stemcells.2007-0131. View