pSpCas9(BB)-2A-Puro (PX459)

CRISPR Human - Deletion NOX4

Experiment
CRISPR Human - Deletion NOX4
Product
pSpCas9(BB)-2A-Puro (PX459) 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

Generation of Knockout cell line with CRISPR/Cas9
Guide RNA sequences for CRISPR/Cas9 were designed at CRISPR design web site (http://crispr.mit.edu/), provided by the Feng Zhang Lab [21]. Insert oligonucleotides for human NOX4 gRNA #1 and #2 are 5’-CACCGGCACATGGGTAAAAGGATG-3’ / 5’-AAACTATCCTTTTACCCATGTGCC-3’ and 5’-CACCGACACTCTTGGCTTACCTCCG-3’ / 5’-AAACCGGAGGTAAGCCAAGAGTGTC-3’, respectively. The NOX4 guide RNA targets the exon 3 of NOX4 gene. The complementary oligonucleotides for guide RNAs (gRNAs) were annealed, and cloned into pX459 CRISPR/Cas9-Puro vector (Addgene, Cambridge, MA). HeLa cells were transfected with either pX459/gRNA #1 or pX459/gRNA #2 using Safectine RU50, according to the manufacturer's instructions. Two days after transfection, cells were treated with 1 μg/ml of puromycin for three days. After two weeks, colonies were isolated with the cloning cylinders, and the NOX4 sequences were analyzed with T7 endonuclease (T7E1) assay, DNA sequencing and Western blot. The antibody for NOX4 was purchased from ProSci (Poway, CA).

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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

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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-Puro (PX459) below.

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Videos

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