Forward Genetic Screening and Single Cell Analysis with CRISPRi Transcriptional Repressors


A Ravanelli1; R Grady1; E Meer2; J Terry2; J Jowdy1
1 Merck KGaA, United States;  2 10x Genomics, UK


Introduction and RelevanceThe power of CRISPR for genome engineering, coupled with the ability to performlarge-scale, whole genome, loss-of-function screening has allowed for newbreakthroughs identifying gene pathways in drug resistance and disease. CRISPR ismost commonly used to create double-stranded breaks that often result in loss ofgene function (CRISPR-KO). However, the full extent of CRISPR’s utility extendsbeyond just targeted cutting of DNA. Nuclease-independent applications of CRISPRprovide all the targeting specificity but for delivery of cargo, such as effectordomains for activation (CRISPRa) or repression (CRISPRi) of target gene expression.CRISPRi allows for targeted inhibition of gene function by delivering transcriptionalrepressor domains to a specific target sequence using modified dCas9+gRNAcomplexes. Gene knockdown is complementary to CRISPR and functional genomicscreening tools. We have developed an optimized CRISPRi system using sequencemodifications and guide design strategies that maximize knockdown efficiency. Wehave also developed 10x Genomics-compatible vectors for single-cell RNAseq.CRISPRi contributes to a powerful toolbox for stand-alone and orthogonalapproaches to discovery and detailed functional genomic analysis atdifferent scales.

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