pX330-U6-Chimeric_BB-CBh-hSpCas9

CRISPR Human - Activation RANKL

Experiment
CRISPR Human - Activation RANKL
Product
pX330-U6-Chimeric_BB-CBh-hSpCas9 from Addgene
Manufacturer
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

Plasmids construction
The pdCas9-Tet1-CD plasmid was constructed based on parental plasmid pX330, which harbored two expression cassettes (a kind gift by Dr Feng Zhang, MIT, USA). Cas9 was also subject to D10A/H840A substitution to construct dCas9, and MS2 RNA elements were inserted into U6 promoter-driven sgRNA cassette at two different sites according to the previous report (briefly designated as sgRNA2.0) [9]. Mouse Tet1-CD was fused to dCas9 domain with a flexible linker (detailed sequences shown below) using Gibson assembly method [28]. MS2 coat protein gene fused with Tet1-CD was inserted into pcDNA3.1/hygro(+) vector (Life Technologies, Carlsbad, CA, USA) using Gibson assembly method. The full amino-acid sequences of dCas9-Tet1 CD and MS2-NLS-Tet1 CD protein were provided in Supplementary Methods. Designed sgRNAs (listed in Supplementary Table S1) were synthesized as oligos (Sangon, Shanghai, China), annealed and cloned into pdCas9-Tet1-CD vector at BbsI digestion sites.

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Reviews

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Discussion

Discussion

1 year 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?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Activation RANKL using pX330-U6-Chimeric_BB-CBh-hSpCas9 from Addgene.

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Manufacturer protocol

Download the product protocol from Addgene for pX330-U6-Chimeric_BB-CBh-hSpCas9 below.

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Videos

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