CRISPR Human - Repression HBV

CRISPR Human - Repression HBV
pX330-U6-Chimeric_BB-CBh-hSpCas9 from Addgene

Protocol tips

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Design and cloning of HBV-specific gRNA/Cas9 plasmids
The candidate 20-base gRNA sequences were derived from the HBV genome sequence of pHBV1.3 (genotype D). We used the CRISPR/Cas9 design tool of the Feng Zhang Laboratory ( crispr.mit.edu ) to search the functional gRNA sequences, which contained the downstream 3′ PAM with GG dinucleotides (N20-NGG). The 5′ 20-base nucleotides were inserted to pX330 and named pX330-U6-HBVgRNA (T n for short). The T mix was mixture of eight pX330-U6-HBVgRNA plasmids in equal amount. The 20-base gRNA of T GFP was derived from the EGFP gene sequence.

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pX330-U6-Chimeric_BB-CBh-hSpCas9 from Addgene has not yet been reviewed for this experiment

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4 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?

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

Paper title
Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome.
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Manufacturer protocol

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

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