lenti dCAS-VP64_Blast

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

Vector design and construction
Vectors used and guide scaffolds will be deposited to Addgene

dCas9-VP64 (Addgene #47319), dCas9-VPR (Addgene #63798), dCas9-VP64 (for SAM activation Addgene # 61425), MCP-p65-hsf1 (Addgene #61426), dCas9-10xGCN4 (Addgene #60903), scFv-VP64 (Addgene #60904), dCas9-p300core (Addgene #61357), dCas9-VP160 (Addgene #48225), Cas9-m4 (Addgene #47316) and MCP-VP647 were previously described.

For all systems tested the original vectors deposited to Addgene were employed allowing us to use the presumably optimal expression vector decided upon by the various depositing laboratories. dCas9-VP64, dCas9-VPR, dCas9-p300, dCas9-VP160, and dCas9-m4 constructs were all driven under CMV promoters. dCas9-VP64 (for SAM activation) and MCP-p65-hsf1 used EF1alpha promoters and dCas9-10xGCN4 and scFv-VP64 used SV40 promoters.

Gibson cloning was utilized to make all variants. For SAM and Scaffold gRNA variants, gene blocks were used (Integrated DNA Technologies). See Supplementary Note 2 for full sequence information.

All dCas9 activator components were cloned into a Drosophila pActin vector using Gibson assembly,34,35 adding a Kozak sequence (GCCACC) immediately upstream of the start codon.

To generate Drosophila MS2-containing sgRNA expression vectors for SAM and Scaffold, the pCFD3 plasmid was modified via Gibson assembly to include the indicated sgRNA tails.36 For all sgRNA plasmids guide oligos were cloned using a BbsI digest.36

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Reviews

Discussion

Papers

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Manufacturer protocol

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Videos

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