Improved vectors and genome-wide libraries for CRISPR screening

NE Sanjana, O Shalem, F Zhang - Nature methods, 2014 - nature.com
Nature methods, 2014nature.com
To the Editor: Genome-wide, targeted loss-of-function pooled screens using the clustered,
regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 in
human and mouse cells provide an alternative screening system to RNA interference (RNAi)
1–4. Previously, we used a genome-scale CRISPR knockout (GeCKO) library to identify loss-
of-function mutations conferring vemurafenib resistance in a melanoma model1. However,
initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell …
To the Editor: Genome-wide, targeted loss-of-function pooled screens using the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi) 1–4. Previously, we used a genome-scale CRISPR knockout (GeCKO) library to identify loss-of-function mutations conferring vemurafenib resistance in a melanoma model1. However, initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, thereby limiting the range of biological systems amenable to screening. We sought to improve both the lentiviral packaging and choice of guide sequences in our original GeCKO library1, where a pooled library of synthesized oligonucleotides was cloned into a lentiviral backbone containing both the Streptococcus pyogenes Cas9 nuclease and the single guide RNA (sgRNA) scaffold. To create a new vector capable of producing higher-titer virus (lentiCRISPRv2), we made the appropriate EvoD version can be applied. Whether the method was properly trained and tested within each conservation stratum is a separate question. Vihinen1 correctly notes that some of the procedural details surrounding EvoD were lacking, prompting Kumar et al. 2 to provide a more thorough description in their response. The additional information supports that EvoD was sensibly calibrated and evaluated.
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