lentiCRISPR v2

CRISPR Human - Deletion EGFP1

CRISPR Human - Deletion EGFP1
lentiCRISPR v2 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.
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

Plasmids encoding sgRNA and Cas9 were purchased from Addgene, and specific sgRNA target sequences were cloned into the CRISPR v2 vector (Addgene plasmid #52961). The 20-bp target sequences of sgRNAs targeting EGFP, AAVS1, and Pten were used previously (–). The 20-bp target sequences of the indicated sgRNAs were as follows: sgEGFP-1, GGGCGAGGAGCTGTTCACCG; sgEGFP-2, GAGCTGGACGGCGACGTAAA; sgAAVS1, GGGGCCACTAGGGACAGGAT; sgNUAK2, TTGATCAGCCCTTCCGCCAG; sgPten, AGATCGTTAGCAGAAACAAA; sg53BP1, CATAATTTATCATCCACGTC. The primers used for PCR amplification of sgRNA target regions were as follows: EGFP-FP, ATGGTGAGCAAGGGCGAGGA; EGFP-RP, TTACTTGTACAGCTCGTCCA; AAVS1-FP, CCCCGTTCTCCTGTGGATTC; AAVS1-RP, ATCCTCTCTGGCTCCATCGT; NUAK2-FP, GCTTTACTGCGCGCTCTGGTACTGC; NUAK2-RP, CAGGCGCCCCGAGCTCTCCC.

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lentiCRISPR v2 from Addgene has not yet been reviewed for this experiment

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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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Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Deletion EGFP1 using lentiCRISPR v2 from Addgene.

Paper title
CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation
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Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR v2 below.

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