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

CRISPR Human - Repression HPV-18 E7

CRISPR Human - Repression HPV-18 E7
pSpCas9(BB)-2A-GFP (PX458) from Addgene

Protocol tips

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 ( 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).

Full paper   Login or join for free to view the full paper.


pSpCas9(BB)-2A-GFP (PX458) from Addgene has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!



3 years ago

Author: Milena Alexeyeva Russian Federation

DNA insert using CRISPR

I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?

Share your thoughts or question with experts in your field by adding a discussion!


Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Repression HPV-18 E7 using pSpCas9(BB)-2A-GFP (PX458) from Addgene.

View full paper   Login or join for free to view the full paper.

Manufacturer protocol

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

Download PDF Download manufacturer protocol


Check out videos that might be relevant for performing CRISPR Human - Repression HPV-18 E7 using pSpCas9(BB)-2A-GFP (PX458) from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms