Wessels, H.-H. et al. Nat. Biotechnol. https://doi.org/10.1038/s41587-020-0456-9 (2020).

Cas13, guided by a single CRIPSR RNA, is known to target RNA and has shown activities in targeted knockdown, RNA editing and RNA imaging without mutating the genome. Cas13d, a subtype of Cas13, has been used for efficient RNA knockdown in mammalian cells. Yet little is known about the design rules of guide RNA (gRNA) for optimal Cas13d activity. To learn more about Cas13d activity, Wessels et al. performed knockdown tiling screens targeting a GFP transgene, as well as endogenous transcripts including cell-surface proteins, in human cells. The researchers evaluated the knockdown efficacy of 24,460 gRNAs and found that both gRNA sequence and target site features affect knockdown efficacy. On the basis of the screen results, they establish an on-target model to predict gRNA knockdown efficacy, which successfully sorted high-activity gRNAs from poorly performing ones. In addition, they provide a resource for optimized gRNAs that target protein-coding transcripts in the human genome.