The advent of genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening has advanced the understanding of molecular systems within cells. Here, we demonstrate the utility of sequentially performed CRISPR knockout screens that use an existing library to explore a biological question across the human genome, and then the remaining cells are used to examine each gene candidate against one common gene of interest. We call this approach "Many vs One" CRISPR screening, made possible by a modified 7SK promoter in place of the U6 promoter to drive expression of a single guide RNA. Inserting this novel 7SK promoter into the ubiquitously used lentiCRISPRv2 backbone is crucial, because it overcomes the need for a substantial increase in CRISPR library coverage during screening, sample processing, and next generation sequencing. This new 7SK vector equals the original lentiCRISPRv2 in lentiviral titer, knockout efficiency, and ease of use.
Keywords: 7SK promoter; CRISPR/Cas9; LentiCRISPR; screening; sequential screen.