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CRISPR: Purify Genomic Loci


Identifying molecules associated with a genomic region of interest in vivo is essential to understanding locus function. Using CRISPR, researchers have expanded chromatin immunoprecipitation (ChIP) to allow purification of any genomic sequence specified by a particular gRNA.

In the enChIP (engineered DNA-binding molecule-mediated ChIP) system, catalytically inactive dCas9 is used to purify genomic DNA bound by the gRNA. An epitope tag(s) can be fused to dCas9 or gRNA for efficient purification. Various epitope tags including 3xFLAG-tag, PA, and biotin tags, can be used for enChIP, as well as an anti-Cas9 antibody. Biotin tagging of dCas9 can be achieved by fusing a biotin acceptor site to dCas9 and co-expressing BirA biotin ligase, as seen in the CAPTURE system. The locus is subsequently isolated by affinity purification against the epitope tag.

Compared to conventional methods for genomic purification, CRISPR-based purification methods are more straightforward and enable direct identification of molecules associated with a genomic region of interest in vivo.

For CRISPR-based purification, design your gRNA sequence to direct dCas9 to a specific locus, avoiding known transcription factor and other protein binding sites.

CRISPR-purify-horizontal3.png


Browse, sort, or search the tables below for CRISPR plasmids to help purify genomic loci.
Plasmids are available for expression in mammalian systems, bacteria, and yeast.


Mammalian

Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
3xFLAG-dCas9/pCMV-7.13xFLAG-dCas9CMV Fujii 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 Aug 11. pii: S0006-291X(13)01329-6. doi: 10.1016/j.bbrc.2013.08.013. Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest. pCMV-7.1
3xFLAG-dCas9/pMXs-puro3xFLAG-dCas9 (Synthetic)LTRPuromycin Fujii Identification of Proteins Associated with an IFNgamma-Responsive Promoter by a Retroviral Expression System for enChIP Using CRISPR. PLoS One. 2014 Jul 22;9(7):e103084. doi: 10.1371/journal.pone.0103084. eCollection 2014. Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest. pMXs-puro
3xFLAG-dCas9/pMXs-IG3xFLAG-dCas9 (Synthetic)LTRenhanced GFP Fujii Identification of Proteins Associated with an IFNgamma-Responsive Promoter by a Retroviral Expression System for enChIP Using CRISPR. PLoS One. 2014 Jul 22;9(7):e103084. doi: 10.1371/journal.pone.0103084. eCollection 2014. Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest. pMXs-IG
3xFLAG-dCas9/pMXs-I23xFLAG-dCas9 (Synthetic)LTRhuman CD2 Fujii Identification of Proteins Associated with an IFNgamma-Responsive Promoter by a Retroviral Expression System for enChIP Using CRISPR. PLoS One. 2014 Jul 22;9(7):e103084. doi: 10.1371/journal.pone.0103084. eCollection 2014. Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest. pMXs-I2
3xFLAG-dCas9/pMXs-neo3xFLAG-dCas9 (Synthetic)LTRNeomycin (select with G418) Fujii Identification of Proteins Associated with an IFNgamma-Responsive Promoter by a Retroviral Expression System for enChIP Using CRISPR. PLoS One. 2014 Jul 22;9(7):e103084. doi: 10.1371/journal.pone.0103084. eCollection 2014. Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest. pMXs-neo
3xFLAG-dCas9/MSCV-EGFP3xFLAG-dCas9 (Synthetic)LTREGFP Fujii Fujii lab 3xFLAG-dCas9 plasmids (unpublished) Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest. MSCV-EGFP
CSII-U6-gRNA-CBh-3xFLAG-PA-dCas9-P2A-PurosgRNA and dCas9 from pX330 (Other)U6 for sgRNA and CBh for dCas9Puromycin Kimura Dr. Sekita CRISPR/Cas9 (unpublished) Lentivirus vector to express guideRNA and dCas9 with puro resistant gene CSII
pLenti_dCas9-2xAMSp-dCas9-2xAM tag (Synthetic), gRNA to be inserted into Bbs I sites (Synthetic)CBh, U6Puromycin Fujii Fujii lab enChIP lentiviral plasmids with 2xAM tag (unpublished) Lentiviral plasmid expressing dCas9-2xAM and gRNA cloned in Bbs I sites CSII-U6-gRNA-CBh-3xFLAG-PA-dCas9-P2A-Puro
pLenti_dCas9-2xAM_hIRF-1Sp-dCas9-2xAM tag (Synthetic), gRNA targeting human IRF-1 promoter (Synthetic)CBh, U6Puromycin Fujii Fujii lab enChIP lentiviral plasmids with 2xAM tag (unpublished) Lentiviral plasmid expressing dCas9-2xAM and gRNA for human IRF-1 promoter CSII-U6-gRNA-CBh-3xFLAG-PA-dCas9-P2A-Puro
pEF1a-FB-dCas9-puroN-terminal Flag and biotin-acceptor-site (FB)-tagged dCas9 (Other), puromycin (Other)Human EF1aPuromycin Xu In Situ Capture of Chromatin Interactions by Biotinylated dCas9. Cell. 2017 Aug 24;170(5):1028-1043.e19. doi: 10.1016/j.cell.2017.08.003. Expresses N-terminal Flag and biotin-acceptor-site (FB)-tagged dCas9 protein pEF1a-FB-puro

Bacteria

Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
3xFLAG-dCas9/p-bacteria3xFLAG-dCas9 (Synthetic)pLtetO-1 Fujii 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 Aug 11. pii: S0006-291X(13)01329-6. doi: 10.1016/j.bbrc.2013.08.013. Expresses 3xFLAG-dCas9 in E. coli in a tetracycline-dependent manner for enChIP analysis to purify specific genomic regions of interest. p15A vector
NM-01Cas9 (Other) Cagney Gerard Cagney unpublished plasmids (unpublished) Bacterial expression of Cas9 with His, MBP, and Strep tags pET21a

Yeast

Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
3xFLAG-dCas9/pTEF1p-CYC1t3xFLAG-dCas9 (Synthetic)TEF1 promoterTRP1 Fujii 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 Aug 11. pii: S0006-291X(13)01329-6. doi: 10.1016/j.bbrc.2013.08.013. Expresses 3xFLAG-dCas9 in budding yeast for enChIP analysis to purify specific genomic regions of interest. p414


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