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 .
Plentirnaguide 001
Manufactured by Addgene
PLentiRNAGuide_001 is a lentiviral vector designed for the expression of single guide RNA (sgRNA) for CRISPR-Cas9 genome editing applications. The vector contains the U6 promoter for sgRNA expression and a puromycin resistance cassette for selection of transduced cells.
Automatically generated - may contain errors
2 protocols using plentirnaguide 001
1
Generating KRAB-dCas9 and FNLS-BE3-SpRY Plasmids
2
Engineered KRAB-dCas9 Repressor Plasmids
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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) (69) 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) (22) .
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 (70) . After gel extraction (Qiagen 28706), we ligated this piece into NheI and KpnI-digested pLentiRNAGuide_001 (Addgene 138150) vector using T4 ligase (NEB M0202M) (71) .
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 (70) . After gel extraction (Qiagen 28706), we ligated this piece into NheI and KpnI-digested pLentiRNAGuide_001 (Addgene 138150) vector using T4 ligase (NEB M0202M) (71) .
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