Casilio-ME

Parent project: [Casilio]

▲From left to right: Albert Cheng, Aziz Taghbalout, [Casilio presentation], Menghan Du, Nathaniel Jillette. Photography by Charles Camarda.

Casilio-ME are DNA methylation editing (ME) modules for Casilio

Project led by Research Scientist, Dr. Aziz Taghbalout.

 

▲Casilio-ME1 mediated activation of DNA methylation silenced genes.

▲Dual delivery of TET and other proteins involved in DNA demethylation.

Publication:

Taghbalout, A., Du, M., Jillette, N., Rosikiewicz, W. Rath, A., Heinen, C., Li, S., Cheng, A.W. (2019) Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways. Nature Communications 10:4296. doi:10.1038/s41467-019-12339-7  PMID: 31541098 PMCID: PMC6754513

Press release:

An epigenetic erase by Mark Wanner [ http://www.jax.org/news-and-insights/2019/september/an-epigenetic-eraser ]

Grant support:

R01HG009900, P30CA034196, Leukemia Research Foundation New Investigator Grant, CA115783

Plasmid listing

Plasmid name/Click for Addgene page Description
pAT1504_pX-SPFE-BbsI(ccdbCam)-5xPBSc Cloning vector for expression of Casilio sgRNAs containing five PUFc-binding sites (PBSc)
pAT1503_pX-SPFE-BbsI(ccdbCam)-5xPBSa Cloning vector for expression of Casilio sgRNAs containing five PUFa-binding sites (PBSa)
pAT1090_PB-EF1a-Hygro_2A_rtTA3-SV40pA_pCW-Flag-PUFa-hTET1(CD) PiggyBac vector for establishement of doxycycline-inducible Casilio-ME1 effector expression cell lines
pAT1502-[PB-4xHSab-EF1a-Blast_2A_rtTA3-SV40pA(-)_pCW-DEST] Destination vector for doxycycline-inducible expression from plasmid hosting piggyBac Blasticidin cassettes
pAT1501-[PB-4xHSab-EF1a-Hygro_2A_rtTA3-SV40pA(-)_pCW-DEST] Destination vector for doxycycline-inducible expression from plasmid hosting piggyBac hygromycin cassettes
pAT1089_PB-EF1a-Blast_2A_rtTA3-SV40pA_pCW-dCas9 PiggyBac vector for establishement of doxycycline-inducible Sp dCas9 expression cell lines
pAT1060_CMV/CAG_Flag-hNEIL2 (R310Q)-PUFa-hTET1(CD) Expression of Flag-tagged Casilio-ME3.1 effector with R310Q NEIL2 mutation hNEIL2(R310-Q)-PUFa-hTET1(CD) in mammalian cells
pAT1059_CMV/CAG_Flag-hNEIL2 (C291S)-PUFa-hTET1(CD) Expression of Flag-tagged Casilio-ME3.1 effector with C291S NEIL2 mutation hNEIL2(C291S)-PUFa-hTET1(CD) in mammalian cells
pAT977_CMV/CAG_Flag-hNEIL2-PUFa-dTET1(CD) Expression of Flag-tagged Casilio-ME3.1 effector containing a catalytically dead hTET1(CD) in mammalian cells
pAT973_CMV/CAG_FlaghGADD45A (G39A, L77E)-PUFa-hTET1(CD) Expression of Flag-tagged Casilio-ME2.2 effector with G39A L77E GADD45A mutations hGADD45A(G39A L77E)-PUFa-hTET1(CD) in mammalian cells
pAT972_CMV/CAG_Flag-hGADD45A (L77E)-PUFa-hTET1(CD) Expression of Flag-tagged Casilio-ME2.2 effector with L77E GADD45A mutation hGADD45A (L77E)-PUFa-hTET1(CD) in mammalian cells
pAT971_CMV/CAG_Flag-hGADD45A (G39A)-PUFa-hTET1(CD) Expression of Flag-tagged Casilio-ME2.2 effector with G39A GADD45A mutation hGADD45A(G39A)-PUFa-hTET1(CD) in mammalian cells
pAT976_CMV/CAG_Flag-hGADD45A-PUFa-dTET1CD Expression of Flag-tagged and catalytically inactive Casilio-ME2.2 effector containing dead hTET1(CD) H1671Y D1673A in mammalian cells
pAT699-CMV/CAG_Flag-hGADD45A-PUFa-hTET1(CD) Expression of Flag-tagged Casilio-ME2.2 effector hGADD45A-PUFa-hTET1(CD) in mammalian cells
pAT596_CMV/CAG_hNEIL2-PUFc Expression of Casilio-ME3.4 effector hNEIL2-PUFc in mammalian cells
pAT595_CMV/CAG_PUFc-hNEIL2 Expression of Casilio-ME3.3 effector PUFc-hNEIL2 in mammalian cells
pAT611_CMV/CAG_PUFa-hNEIL2-hTET1(CD) Expression of Casilio-ME3.2 effector PUFa-hNEIL2-hTET1(CD) in mammalian cells
pAT608_CMV/CAG_Flag-hNEIL2-PUFa-hTET1(CD) Expression of Flag-tagged Casilio-ME3.1 effector hNEIL2-PUFa-hTET1(CD) in mammalian cells
pAT892_CMV/CAG_PUFa-p65HSF1 Expression of Casilio activator PUFa-p65HSF1 in mammalian cells
pAT356-CMV/CAG_hGADD45A-PUFc Expression of Casilio-ME2.4 effector hGADD45A-PUFc in mammalian cells
pAT355_CMV/CAG_PUFc-hGADD45A Expression of Casilio-ME2.3 effector PUFc-hGADD45A in mammalian cells
pAT635_CMV/CAG_hGADD45A-PUFa-hTET1(CD) Expression of untagged Casilio-ME2.2 effector hGADD45A-PUFa-hTET1(CD) in mammalian cells
pAT360_CMV/CAG_PUFa-hGADD45A-hTET1(CD) Expression of untagged Casilio-ME2.1 effector PUFa-hGADD45A-hTET1(CD) in mammalian cells
pAT341_CMV/CAG_PUFa-TET1CD (H1671Y D1673A) (dead TET1) Expression of catalytically inactive Casilio-ME1 effector containing dead hTET1(CD) H1671Y D1673A in mammalian cells
pAT817_CMV/CAG_TALE_F-hTET1(CD) Expression of TALE-hTET1(CD) in mammalian cells targeting MLH1 promoter region
pAT806_CMV/CAG_TALE_D-hTET1(CD) Expression of TALE-hTET1(CD) in mammalian cells targeting MLH1 promoter region
pAT812_CMV/CAG_TALE_B-hTET1(CD) Expression of TALE-hTET1(CD) in mammalian cells targeting MLH1 promoter region
pAT801_CMV/CAG_TALE_A-hTET1(CD) Expression of TALE-hTET1(CD) in mammalian cells targeting MLH1 promoter region
pAT890_CMV/CAG_dCas9-hTET1(CD) Expression of Sp dCas9-hTET1(CD) fusion protein in mammalian cells
pAT888_CMV/CAG_PUFa-hTET1(CD) Expression of untagged Casilio-ME1 effector PUFa-hTET1(CD) in mammalian cells
pAT243_pX-sgRNA-5xPBSa-5xPBSc Cloning vector for expression of sgRNAs with dual PUF-Binding Sites (5xPBSa – 5xPBSc) of Casilio-ME 2.3, 2.4, 3.3 and 3.4