To construct PE plasmid, PE cassette was amplified from pCMV-PE2 (Addgene no.132775) and amplified product was inserted into pBAtC (Addgene no.78097), generating pBAtC-NGG-PE2 vector. To build NG-PAM targetable PE vector, we introduced same mutations with pX330-SpCas9-NG (Addgene no.117919) in our Cas9 fragment. For introducing pegRNA cassette, oligos representing the target sequences, sgRNA scaffold and 3’ extensions were annealed and cloned into pRG2 vector (Addgene no. 104174) with additional AtU6-26 promoter using BsaI to build AtU6-26p-pegRNA vector. Restriction enzyme-digested fragment encoding AtU6-26p-pegRNA cassette was inserted into pBAtC-NG-PE2 vector digested with same restriction enzyme. To construct nicking sgRNA cassette, oligos representing nicking sequences were annealed and cloned into AarI-digested PE plasmid. Oligos used for preparing plasmid was designed using Cas-designer (22 (link)) and PE-designer (23 (link)).
Prg2 vector
Manufactured by Addgene
The PRG2 vector is a plasmid that can be used for gene expression studies in mammalian cells. It contains a promoter sequence that drives the expression of the gene of interest. The core function of the PRG2 vector is to facilitate the cloning and expression of target genes in cellular systems.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using prg2 vector
1
Generating NG-PAM Targetable PE Vectors
2
CRISPR-Cas9 Genome Editing Vectors
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The Streptococcus pyogenes Cas9 sequence [38 (link)] and the designed promiscuous sgRNA sequences that target Albumin and PCSK9 were cloned into the AAV plasmid backbone used in a previous study [39 (link)] to create Cas9 (pAAV-Cas9) and sgRNA (pAAV-Albumin and pAAV-PCSK9) expression vectors. Cas9 expression is under the control of the CMV promoter and sgRNA expression is under the control of the U6 promoter. Guide sequences targeting FANCF, VEGFA, and HBB genes [14 (link)] were cloned into pRG2 vector (Addgene #104174).
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