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

  1. 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
  2. GeCKO Library v1 and v2 transformation protocol : PDF, benchling
  3. Readout primers for Illumina sequencing of sgRNA cassette : MS Excel
  4. 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)
  5. 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.

oligo protocol previewlibrary protocol preview