pLX_311-Cas9

CRISPR Human - Repression AKT2

Experiment
CRISPR Human - Repression AKT2
Product
pLX_311-Cas9 from Addgene
Manufacturer
Addgene

Protocol tips

Protocol tips
here 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

CRISPR-Cas9 screening
Cancer cell lines were transduced with a lentiviral vector expressing the Cas9 nuclease under blasticidin selection (pXPR-311Cas9). Each Cas9 expressing cell line was subjected to a Cas9 activity assay (see below) to characterize the efficacy of CRISPR-Cas9 in these cell lines (Table S1). Cell lines with less than 45% measured Cas9 activity were considered ineligible for screening. Stable polyclonal Cas9+ cell lines were then infected in replicate (n = 3) at low multiplicity of infection (MOI<1) with a library of 123,411 unique sgRNAs targeting 19,050 genes (6 sgRNAs per gene), 1,864 miRNAs and 1,000 non-targeting control sgRNAs (GeCKO v2), selected in puromycin and blasticidin for 7 days and then passaged without selection while maintaining a representation of 500 cells per sgRNA until a defined time point. Genomic DNA was purified from end cell pellets and the guide sequence PCR amplified with sufficient gDNA to maintain representation, and quantified using massively parallel sequencing.

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Reviews

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Discussion

Discussion

5 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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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Repression AKT2 using pLX_311-Cas9 from Addgene.

Paper title
Genomic copy number dictates a gene-independent cell response to CRISPR-Cas9 targeting
Full paper
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Manufacturer protocol

Download the product protocol from Addgene for pLX_311-Cas9 below.

Download PDF Download manufacturer protocol

Videos

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