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Pcc 09

Manufactured by Addgene
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PCC_09 is a laboratory equipment product. It serves as a core function to facilitate specific tasks or processes in a laboratory environment. No further details about its intended use or capabilities are provided.

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Lab products found in correlation

Lenticas9 blast, by Addgene (2 mentions) Plentirnaguide 001, by Addgene (2 mentions) Plenti fnls p2a puro, by Addgene (1 mentions) Pcag cbe4max spry p2a egfp, by Addgene (1 mentions) T4 ligase, by New England Biolabs (1 mentions)

2 protocols using pcc 09

1

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) .
Morris J., Daniloski Z., Domingo J., Barry T., Ziosi M., Glinos D.A., Hao S., Mimitou E.P., Smibert P., Roeder K., Katsevich E., Lappalainen T., & Sanjana N.E. (2021). Discovery of target genes and pathways of blood trait loci using pooled CRISPR screens and single cell RNA sequencing.
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2

Generating KRAB-dCas9 and FNLS-BE3-SpRY Plasmids

Cited 2 times
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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) (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.
Talon J., Salvatore M., O'Neill R.E., Nakaya Y., Zheng H., Muster T., García-Sastre A, & Palese P. (2000). Influenza A and B viruses expressing altered NS1 proteins: A vaccine approach. Proceedings of the National Academy of Sciences of the United States of America, 97(8), 4309-4314.
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