MLM3636

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

Construction of sgRNA expression plasmids
Pairs of DNA oligonucleotides encoding the variable 20 nt sgRNA targeting sequences were annealed together to generate short double-strand DNA fragments with 4bp overhangs (Supplementary Table 2). These fragments were ligated into BsmBI-digested plasmid pMLM3636 to yield DNA encoding a chimeric +103 single-chain guide RNA3,5 under the expression of a human U6 promoter. The pMLM3636 plasmid and its full DNA sequence are available from Addgene (http://www.addgene.org/crispr-cas).

Construction of dCas-VP64 expression plasmids
DNA encoding the Cas9 nuclease harboring inactivating D10A/H840A mutations (dCas9) was amplified by PCR from plasmid pMJ841 (Addgene plasmid #39318) using primers that add a T7 promoter site 5’ to the start codon and a nuclear localization signal at the carboxy-terminal end of the Cas9 coding sequences and cloned into a plasmid containing a CMV promoter as previously described3 to yield plasmid pMLM3629. Oligonucleotides encoding a triple FLAG epitope were annealed and cloned into XhoI and PstI sites in plasmid pMLM3629 to generate plasmid pMLM3647 expressing dCas9 with a C-terminal flag FLAG tag. DNA sequence encoding a Gly4Ser linker followed by the synthetic VP64 activation domain was introduced downstream of the FLAG-tagged dCas9 in plasmid pMLM3647 to yield plasmid pSL690. The D10A/H840A mutations were also introduced by QuikChange site-directed mutagenesis (Agilent) into plasmid pJDS246, which encodes a FLAG-tagged Cas9 sequence that has been codon optimized for expression in human cells, to yield plasmid pMLM3668. DNA sequence encoding the Gly4Ser linker and the VP64 activation domain were then cloned into pMLM3668 to yield a codon-optimized dCas9-VP64 expression vector named pMLM3705. The full amino acid sequence of the dCas9-VP64 protein is shown in Supplementary Figure 6.

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Manufacturer protocol

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