pAC2-dual-dCas9VP48-sgExpression

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Cloning
A two-step fusion PCR was performed to amplify Cas9 Nickase ORF without stop codon from the pX335 vector (Addgene: 42335), incorporate H840A mutation, EcoRI-AgeI restriction site on the 5′ end as well as an FseI site on the 3′ end (EcoRI-AgeI-dCas9-FseI fragment). The 3× minimal VP16 activation domain coding fragment (VP48) was excised from a vector (Addgene: 20342) containing NLSM2rtTA coding sequence by FseI and EcoRI digestion (FseI-TA-EcoRI fragment). The two fragments were ligated into pCR8/GW/TOPO (Invitrogen) vector digested by EcoRI to generate a gateway compatible dCas9VP48 coding plasmid. The dCas9VP48 coding sequence was subsequently excised and cloned into pX355 vector (Addgene: 42335) by AgeI-EcoRI digestion to replace dCas9 Nickase to create a chimeric vector that expresses both the dCas9VP48 and the sgRNA (dCas9VP48-U6-sgRNA-chimeric). sgRNA spacers were cloned into the BbsI-digested vector by annealing oligos as previously described14. For construction of dCas9VP160, a gBlocks gene fragment containing coding sequence for 10 tandem repeats of VP16 domains separated by Glycine-Serine (GS) linker was ordered from Integrated DNA Technology (IDT) and amplified by PCR primers containing FseI and EcoRI sites to replace VP48 fragment in pCR8-dCas9VP48 to generate pCR8-dCas9VP160. A pmax-DEST gateway destination vector was constructed by replacing GFP coding sequence in pmaxGFP (Clontech) by a gateway destination cassette (Invitrogen). The pCR8-dCas9VP160 vector was then recombined with pmax-DEST via LR clonase-mediated recombination to create pmax-dCas9VP160 expression plasmid. For the endogenous gene experiments, sgRNAs were cloned by oligo cloning method mentioned above into a PBneo-sgRNA expression vector sgRNA target sequences, oligos for cloning are listed in Supplementary information, Table S1. Plasmids are deposited on Addgene and additional information is available at http://www.crispr-on.org



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Manufacturer protocol

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