lentiCRISPR v2

CRISPR Human - Repression PCSK9

CRISPR Human - Repression PCSK9
lentiCRISPR v2 from 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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lentiCRISPR v2 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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Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR v2 below.

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