lentiCRISPR v2

Protocol tips

Upstream tips	Protocol tips	Downstream 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.

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

Generation of Knockout Cells Using CRISPR/Cas9 Genome Editing
pSpCas9(BB)-2A–Puro (PX459; Addgene plasmid #48139) and lentiCRISPRv2 (Addgene plasmid #52961) were gifts from Dr. Feng Zhang (Ran et al., 2013; Sanjana et al., 2014). The 20 nucleotide guide sequences were designed using the CRISPR design tool at http://www.genome-engineering.org/crispr or were taken from archived guide sequences from the Genome-scale CRISPR knock-out (GeCKO2) library (Sanjana et al., 2014). Single guide RNAs (sgRNAs) were cloned into PX459 expression vector containing the human codon-optimized Cas9. HEK293A cells were transfected using PolyJet DNA in vitro Transfection Reagent according to the manufacturer’s instructions. In addition, sgRNAs were cloned into the lentiviral vector lentiCRISPRv2. Lentivirus was prepared in 293T cells, and the viral supernatant was collected, and added to MEFs or 3T3-L1 cells with polybrene. 16 hr after infection, the media was changed. 24 hr post transfection, cells were selected with puromycin for 2–3 days. Following removal of puromycin, cells were allowed to recover in regular growth media for 24 hr before being single-cell sorted by FACs (UCSD; Human Embryonic Stem Cell Core, BDInflux) into 96-well plate format. Single clones were expanded and screened by protein immunoblotting, genomic sequencing, and functional assays.

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Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR v2 below.

Download manufacturer protocol

Videos

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