In order to use the lentiCRISPR plasmid to knock out individual genes you will first need to clone in a spacer sequence to target your gene of interest (Protocol 1 below). To use the GeCKO library for genome-wide screens you will first need to re-transform the GeCKO library DNA that we sent you in order to get more DNA that will be sufficient for virus production (Protocol 2 below). Since the pooled library requires a significantly different amplification than standard plasmids, please read the entire protocol before starting GeCKO library amplification. Each protocol provides step-by-step instructions with an overview of the vectors (available at Addgene), sgRNA design and at-the-bench cloning/DNA amplification.
List of protocols and primers
- Protocol to clone custom sgRNAs into lentiCRISPR, lentiCRISPRv2, or lentiGuide-Puro : PDF, benchling
- Detailed instructions on design of oligos for your sgRNA, digestion of backbone vector, and ligation
- GeCKO Library v1 and v2 transformation protocol : PDF, benchling
- Readout primers for Illumina sequencing of sgRNA cassette : MS Excel
- GeCKO library indexes (sgRNA sequences and genes targeted) in comma-separated text: (open in R, Matlab or text editor…. please avoid saving in Excel since it will reformat some gene names as calendar dates)
- Lentivirus production and viral titer quantification protocols from the Broad GPP (GPP website)
Questions? Anything unclear? Ask away on the CRISPR Discussion Forum. Clicking on the previews below will download a high-res, PDF version.