PurposeExpression of human codon-optimized dCas9-DNMT3A fusion with T2A-PuroR (v2) for targeted DNA methylation in mammalian cells; cloning backbone for sgRNA
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||74407||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 4246
- Total vector size (bp) 10152
Vector typeMammalian Expression, CRISPR
Growth in Bacteria
Gene/Insert nameS. pyogenes dCas9 fused with the catalytic domain of human DNMT3A (amino acids P602-V912) and T2A-PuroR_v2
SpeciesH. sapiens (human), Synthetic; S. pyogenes
Insert Size (bp)5906
MutationD10A and H840A in S.pyogenes Cas9
GenBank IDAKA60242.1 NP_072046.2
Entrez GeneDNMT3A (a.k.a. DNMT3A2, M.HsaIIIA, TBRS)
- Promoter CBh
/ Fusion Proteins
- 3xFLAG (N terminal on insert)
- SV40 NLS (N terminal on insert)
- T2A-PuroR (C terminal on insert)
- Cloning method Restriction Enzyme
- 5′ cloning site BamHI (not destroyed)
- 3′ cloning site FseI (not destroyed)
- 5′ sequencing primer GS-linker For 5' GAAGAGGTACACCAGCACCAAAG 3'
- 3′ sequencing primer BGH Rev (Common Sequencing Primers)
The catalytic domain of human DNMT3A (amino acids P602-V912) was derived from the plasmid pcDNA3/Myc-DNMT3A (Addgene, plasmid #35521) (Chen et al., 2005, J Cell Biochem 95: 902-917). Undesired BbsI restriction site was removed by site-directed mutagenesis, without affecting the amino acid sequence.
Plasmid pSpCas9n(BB)-2A-Puro (PX462) (Addgene, plasmid #48141) (Ran et al., 2013, Nat Protoc 8: 2281-2308) was used as a backbone. Additional H840A mutation was introduced into Cas9n D10A nickase. Puromycin gene was mutated by site-directed mutagenesis (H166R) to improve its efficacy.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pdCas9-DNMT3A-PuroR_v2 was a gift from Vlatka Zoldoš (Addgene plasmid # 74407 ; http://n2t.net/addgene:74407 ; RRID:Addgene_74407)
For your References section:Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M, Zoldos V. Nucleic Acids Res. 2016 Mar 11. pii: gkw159. 10.1093/nar/gkw159 PubMed 26969735