pSpCas9(BB)-2A-GFP (PX458)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

CRISPR/Cas9 constructs and sgRNA design.
Two pairs of sgRNAs were designed by using the ZiFit Web application (http://zifit.partners.org/) to target the amino-terminal regions of the HPV-18 E6 and E7 open reading frames (ORFs). RGNs were constructed by cloning HPV-specific sgRNAs into the px330 vector (Addgene) expressing S. pyogenes Cas9 (16). sgRNAs were also cloned into the px458 vector, an alternative version of px330 containing a gfp marker useful for flow cytometric analysis (22). RGN function was tested by generating a vector containing either HPV-18 E6- or E7-derived viral DNA targets inserted in frame between an HIV-1 rev gene fragment encoding amino acids 1 to 59 of Rev (23) and a 3′ gfp indicator gene. Following cotransfection of the reporter plasmid with a S. pyogenes Cas9/sgRNA expression construct, function was determined by detecting the specific loss of Rev and green fluorescent protein (GFP) expression by Western blotting and flow cytometry, respectively. HPV-16-specific sgRNAs targeting the HPV-16 E6 and E7 ORFs integrated into the SiHa cell line were designed and tested by using a similar approach. The following gene-specific sgRNA sequences were used and constructed, as outlined previously (16): HPV-18 E6t1 (GGCGCTTTGAGGATCCAACA), HPV-18 E6t2 (GAAGCTACCTGATCTGTGCA), HPV-18 E7t1 (GGAGCAATTAAGCGACTCAG), HPV-18 E7t2 (GAAGAAAACGATGAAATAGA), HPV-16 E6t1 (GCAACAGTTACTGCGACGTG), and HPV-16 E7t1 (GCCAGCTGGACAAGCAGAAC) (nucleotides in boldface type indicate mismatched 5′ G residues required for transcription initiation from a U6 promoter).

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-GFP (PX458) below.

Download manufacturer protocol

Videos

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