Massively parallel Cas13 screens reveal principles for guide RNA design

Nat Biotechnol. 2020 Jun;38(6):722-727. doi: 10.1038/s41587-020-0456-9. Epub 2020 Mar 16.

Abstract

Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knockdown without altering the genome. To define rules for the design of Cas13d guide RNAs (gRNAs), we conducted massively parallel screens targeting messenger RNAs (mRNAs) of a green fluorescent protein transgene, and CD46, CD55 and CD71 cell-surface proteins in human cells. In total, we measured the activity of 24,460 gRNAs with and without mismatches relative to the target sequences. Knockdown efficacy is driven by gRNA-specific features and target site context. Single mismatches generally reduce knockdown to a modest degree, but spacer nucleotides 15-21 are largely intolerant of target site mismatches. We developed a computational model to identify optimal gRNAs and confirm their generalizability, testing 3,979 guides targeting mRNAs of 48 endogenous genes. We show that Cas13 can be used in forward transcriptomic pooled screens and, using our model, predict optimized Cas13 gRNAs for all protein-coding transcripts in the human genome.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Computational Biology / methods*
  • Gene Editing / methods*
  • Gene Knockdown Techniques / methods*
  • HEK293 Cells
  • Humans
  • RNA, Guide, CRISPR-Cas Systems
  • Sequence Analysis, RNA

Substances

  • RNA, Guide, CRISPR-Cas Systems