lentiCRISPR v2

CRISPR Mouse - Repression Mstn

Experiment
CRISPR Mouse - Repression Mstn
Product
lentiCRISPR v2 from Addgene
Manufacturer
Addgene

Protocol tips

Protocol tips
There is no need to perform a negative control golden-gate reaction (without
insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

Mefferd et al. (2015) reported recently that expression of sgRNAs can be driven by small tRNA promoters (∼70 bp), making it possible to express two full-length sgRNAs using only ∼350 bp of space, about the size of one sgRNA-expressing cassette with a U6 promoter. We tested the potential of this approach by first comparing side-by-side the genome editing efficiency of CRISPR/Cas9 with U6 promoter- and tRNA promoter-driven sgRNA expression, respectively. For efficient delivery of CRISPR materials to cells in vitro, we reconstructed the original lentiCRISPR v2 for Streptococcus pyogenes (SpCas9) targeting (Sanjana et al. 2014) to make it adaptable for SaCas9 targeting by switching the Cas9 and sgRNA cassettes and named the new vector lentiSaCRISPR v2. We then made three versions of lentiSaCRISPR v2 constructs with different sgRNA promoters—U6pro, tRNAGLNpro, and tRNAPROpro—with the latter two tRNA promoters reported to be able to effectively drive the expression of sgRNAs in SaCRISPR mediated targeting (Fig. 1A; Mefferd et al. 2015). We next made constructs with different lentiSaCRISPR vectors targeting several mouse genes as well as a human gene, using previously screened sgRNA sequences that had shown high on-target efficiency with a U6 promoter.

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Reviews

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Discussion

Discussion

4 years ago

Author: Mario Udinese Italy

Floxing mice with CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Discussion

5 years ago

Author: Ben Saar Israel

How to choose a region to target for CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

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

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Mouse - Repression Mstn using lentiCRISPR v2 from Addgene.

Paper title
CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter
Full paper
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Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR v2 below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Mouse - Repression Mstn using lentiCRISPR v2 from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

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