pLX_311-Cas9

Protocol tips

Upstream tips	Protocol tips	Downstream 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.

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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Papers

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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