To generate the KRAB-dCas9 (lentiCRISPRi(v1)-Blast) and KRAB-dCas9-MeCP2 (lentiCRISPRi(v2)-Blast) plasmids, KRAB and dCas9 were PCR amplified from pCC_09 (Addgene 139094) (92 (link)) and the MeCP2 effector domain was synthesized as a gBlock (IDT). KRAB and MeCP2 were linked to dCas9 with flexible glycine-serine linkers and cloned into lentiCas9-Blast (Addgene 52962) (23 (link)). To generate the FNLS-BE3-SpRY (lentiBE3-SpRY-Blast) plasmid, we used Gibson cloning to replace the puromycin resistance gene in pLenti-FNLS-P2A-Puro (Addgene 110841) with blasticidin resistance from lentiCRISPRi(v2)-Blast. We then used Gibson cloning to replaced SpCas9(D10A) with the SpRY nickase from pCAG-CBE4max-SpRY-P2A-EGFP (Addgene 139999) (51 (link)). To generate the gRNA vector (lentiGuideFE-Puro), we digested pCC_09 with NheI and KpnI to isolate the U6 promoter and Cas9 guide RNA scaffold with the F+E scaffold modification (93 (link)). After gel extraction (Qiagen 28706), we ligated this piece into NheI and KpnI-digested pLentiRNAGuide_001 (Addgene 138150) vector using T4 ligase (NEB M0202M) (94 (link)). Primer sequences for Gibson cloning reactions are available in Table S1F .
Protocol detail
Generating KRAB-dCas9 and FNLS-BE3-SpRY Plasmids
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Corresponding Organization : KTH Royal Institute of Technology
Other organizations : Carnegie Mellon University, University of Pennsylvania
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Variable analysis
independent variables
- KRAB and dCas9 PCR amplified from pCC_09
- MeCP2 effector domain synthesized as a gBlock
- Replacement of puromycin resistance gene in pLenti-FNLS-P2A-Puro with blasticidin resistance from lentiCRISPRi(v2)-Blast
- Replacement of SpCas9(D10A) with the SpRY nickase from pCAG-CBE4max-SpRY-P2A-EGFP
- Generation of KRAB-dCas9 (lentiCRISPRi(v1)-Blast) plasmid
- Generation of KRAB-dCas9-MeCP2 (lentiCRISPRi(v2)-Blast) plasmid
- Generation of FNLS-BE3-SpRY (lentiBE3-SpRY-Blast) plasmid
- Generation of gRNA vector (lentiGuideFE-Puro)
- Lentiviruses used to deliver plasmids (lentiCRISPRi(v1)-Blast, lentiCRISPRi(v2)-Blast, lentiBE3-SpRY-Blast, lentiGuideFE-Puro)
- PCC_09 (Addgene 139094)
- LentiCas9-Blast (Addgene 52962)
- PLenti-FNLS-P2A-Puro (Addgene 110841)
- PCAG-CBE4max-SpRY-P2A-EGFP (Addgene 139999)
- PLentiRNAGuide_001 (Addgene 138150)
- Not explicitly mentioned
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