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Fujii Lab CRISPR Plasmids Available from Addgene


Originally developed in 2009 (Hoshino & Fujii), the locus-specific ChIP technologies enable us to purify specific genomic regions retaining molecular interactions and identify molecules interacting with those regions. Locus-specific ChIP technologies consist of insertional chromatin immunoprecipitation (iChIP) and engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP; Fujita & Fujii, 2013).

In iChIP, specific genomic regions tagged with the recognition sequences of an exogenous DNA-binding protein such as LexA are subjected to affinity purification (Fig. 1). In enChIP, specific genomic regions are tagged with engineered DNA-binding molecules such as TAL proteins and the CRISPR system consisting of a catalytically inactive form of Cas9 (dCas9) plus guide RNA (gRNA) for biochemical purification (Fig. 2).

For additional information and protocols, check our references for the plasmids or browse the Fujii Lab web page.

Figure 1: iChIP general scheme

Fujii lab iChIP cartoon

Figure 2: enChIP general scheme

enChIP-cartoon

Fujii Lab Plasmids

Individual plasmids can be ordered via the links below:

  ID Plasmid Description
47948 3xFLAG-dCas9/pCMV-7.1 enChIP plasmid, expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest
48807 8xLexA-binding elements/pMD20 iChIP plasmid, LexA-binding elements
48874 3xFNLDD/pCMV-7.1 iChIP plasmid, expresses 3xFNLDD in mammalian cells
48880 3xFNLDD/pMXs-I2 iChIP plasmid, expresses 3xFNLDD in mammalian cells
48972 3xFNLDD/pMXs-IG iChIP plasmid, expresses 3xFNLDD in mammalian cells
49536 3xFNLDD/pMXs-puro iChIP plasmid, expresses 3xFNLDD in mammalian cells
51240 3xFLAG-dCas9/pMXs-puro Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest
51258 3xFLAG-dCas9/pMXs-IG Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest
51259 3xFLAG-dCas9/pMXs-I2 Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest
51260 3xFLAG-dCas9/pMXs-neo Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest
61079 gRNA-hIRF-1 #12 Expresses a guide RNA (gRNA) to target human IRF-1 promoter
61080 gRNA-hIRF-1 #12/pSIR Expresses a guide RNA (gRNA) to target human IRF-1 promoter
62190 3xFLAG-dCas9/pTEF1p-CYC1t Expresses 3xFLAG-dCas9 in budding yeast for enChIP analysis to purify specific genomic regions of interest
63589 3xFN-Tel-TAL/pCMV-7.1 Expresses a 3xFLAG-tagged TAL protein recognizing a telomere repeat
63590 3xFN-Tel-TAL/pMXs-puro Expresses a 3xFLAG-tagged TAL protein recognizing a telomere repeat. A retroviral expression vector with the puromycin resistance gene
63591 3xFN-Tel-TAL/pMXs-neo Expresses a 3xFLAG-tagged TAL protein recognizing a telomere repeat. A retroviral expression vector with the neomycin resistance gene
64325 3xFLAG-dCas9/p-bacteria Expresses 3xFLAG-dCas9 in E. coli in a tetracycline-dependent manner for enChIP analysis to purify specific genomic regions of interest
82613 3xFLAG-dCas9/MSCV-EGFP Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest
85586 gRNA Cloning Vector Bbs I ver. 2 An empty sgRNA expression vector. sgRNA sequence can be cloned into BbsI site. gRNA scaffold has efficient ver. 2 structure.

References

  • Identification of proteins associated with an IFNgamma-responsive promoter by a retroviral expression system for enChIP using CRISPR. Fujita T, Fujii H. PLoS One. 2014 Jul 22;9(7):e103084. doi: 10.1371/journal.pone.0103084.PubMed.

  • Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Fujita T, Fujii H. Biochem. Biophys. Res. Commun. 2013 Sep 13;439(1):132-6. doi: 10.1016/j.bbrc.2013.08.013.PubMed.

  • Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Fujita T, Asano Y, Ohtsuka J, Takada Y, Saito K, Ohki R, Fujii H. Sci Rep. 2013 Nov 8;3:3171. doi: 10.1038/srep03171.PubMed.

  • Direct identification of insulator components by insertional chromatin immunoprecipitation. Fujita T, Fujii H. PLoS One. 2011;6(10):e26109. doi: 10.1371/journal.pone.0026109.PubMed.

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