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Pguide

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PGuide is a product offered by Addgene, designed to assist researchers in the organization and storage of their plasmids. It provides a structured guide for labeling and cataloging plasmid samples.

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Lab products found in correlation

Px330 u6 chimeric bb cbh hspcas9, by Addgene (2 mentions) Premade adenovirus type 5 particles, by Vector Biolabs (2 mentions) Pcas9 gfp, by Addgene (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions)

3 protocols using pguide

1

Adenoviral Constructs for CRISPR Studies

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The BE3-encoding gene and synthetic polyadenylation sequence from pCMV-BE3, the CAG reporter from pCas9_GFP (Addgene plasmid #44719), and the U6 promoter-driven gRNA cassette from pGuide (Addgene plasmid #64711) with the protospacer sequence 5’-CAGGTTCCATGGGATGCTCT-3’ (for Pcsk9 studies), the protospacer sequence 5’-CATTCAACGTCACAACCACC-3’ (for the Hpd studies), or the protospacer sequence 5’-GGTGCTAGCCTTGCGTTCCG-3’ (control studies: irrelevant protospacer not matching any sequence in the mouse genome) were cloned into pDUAL-Basic expression vector. For R26mTmG/+ experiments, which used SpCas9 and not the BE3, the mTmG protospacer (5’-ATTATACGAAGTTATATTAA-3’) was cloned into plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (a gift from Feng Zhang; Addgene plasmid # 42230). Vector Biolabs (Malvern, PA) used these constructs to generate recombinant adenovirus type 5 particles. Premade adenovirus type 5 particles containing the GFP transgene or Cre recombinase under a CMV promoter were obtained from Vector Biolabs. Ad viral vectors are referred to as Ad.BE3.Pcsk9, Ad.BE3.Hpd, Ad.BE3.Null, Ad.SpCas9.mTmG, Ad.GFP, and Ad.Cre, and the titers are indicated in Supplementary Table 2.
Rossidis A.C., Stratigis J.D., Chadwick A.C., Hartman H.A., Ahn N.J., Li H., Singh K., Coons B.E., Li L., Lv W., Zoltick P.W., Alapati D., Zacharias W., Jain R., Morrisey E.E., Musunuru K, & Peranteau W.H. (2018). In utero CRISPR-mediated therapeutic editing of metabolic genes. Nature medicine, 24(10), 1513-1518.
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2

CRISPR gRNA Construction and Validation

Cited 1 time
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Using the web tool CRISPOR,19 (link) gRNA spacer sequences were selected to target sites in the PCSK9, ANGPTL3, and AGT promoters, prioritizing sequences with high MIT specificity scores (Supplemental Table 1). Each spacer’s sense and antisense oligonulceotide strands were annealed and ligated into a gRNA expression vector (pGuide, Addgene plasmid #64711). After transformations into competent E. coli cells (Takara Bio, catalog #636763), colonies were picked, cultured, and miniprepped using Mini Spin Columns (Epoch Life Science, catalog #1910250). DNA preparations were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Synthetic gRNAs were obtained from Integrated DNA Technologies.
Whittaker M.N., Testa L.C., Quigley A., Jindal I., Cortez-Alvarado S.V., Qu P., Yang Y., Alameh M.G., Musunuru K, & Wang X. (2023). Epigenome Editing Durability Varies Widely Across Cardiovascular Disease Target Genes. bioRxiv.
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3

Adenoviral Constructs for CRISPR Studies

Check if the same lab product or an alternative is used in the 5 most similar protocols
The BE3-encoding gene and synthetic polyadenylation sequence from pCMV-BE3, the CAG reporter from pCas9_GFP (Addgene plasmid #44719), and the U6 promoter-driven gRNA cassette from pGuide (Addgene plasmid #64711) with the protospacer sequence 5’-CAGGTTCCATGGGATGCTCT-3’ (for Pcsk9 studies), the protospacer sequence 5’-CATTCAACGTCACAACCACC-3’ (for the Hpd studies), or the protospacer sequence 5’-GGTGCTAGCCTTGCGTTCCG-3’ (control studies: irrelevant protospacer not matching any sequence in the mouse genome) were cloned into pDUAL-Basic expression vector. For R26mTmG/+ experiments, which used SpCas9 and not the BE3, the mTmG protospacer (5’-ATTATACGAAGTTATATTAA-3’) was cloned into plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (a gift from Feng Zhang; Addgene plasmid # 42230). Vector Biolabs (Malvern, PA) used these constructs to generate recombinant adenovirus type 5 particles. Premade adenovirus type 5 particles containing the GFP transgene or Cre recombinase under a CMV promoter were obtained from Vector Biolabs. Ad viral vectors are referred to as Ad.BE3.Pcsk9, Ad.BE3.Hpd, Ad.BE3.Null, Ad.SpCas9.mTmG, Ad.GFP, and Ad.Cre, and the titers are indicated in Supplementary Table 2.
Rossidis A.C., Stratigis J.D., Chadwick A.C., Hartman H.A., Ahn N.J., Li H., Singh K., Coons B.E., Li L., Lv W., Zoltick P.W., Alapati D., Zacharias W., Jain R., Morrisey E.E., Musunuru K, & Peranteau W.H. (2018). In utero CRISPR-mediated therapeutic editing of metabolic genes. Nature medicine, 24(10), 1513-1518.
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