High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation

Cell Rep. 2020 Mar 3;30(9):2859-2868.e5. doi: 10.1016/j.celrep.2020.02.010.

Abstract

A key limitation of the widely used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG), but their activity has been measured at only a small number of endogenous sites. Here, we devise a high-throughput Cas9 pooled competition screen to compare the performance of Cas9 variants at thousands of genomic loci for gene knockout, transcriptional activation, and inhibition. We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cleavage. Of the PAM-flexible variants, we find that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combine xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants.

Keywords: CRISPRa; CRISPRi; Cas9; PAM flexible; mutagenesis; pooled CRISPR screens; protospacer-adjacent motif; xCas9-NG.

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-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • Endonucleases / metabolism
  • Gene Knockout Techniques*
  • Genetic Variation*
  • HEK293 Cells
  • High-Throughput Screening Assays*
  • Humans
  • INDEL Mutation / genetics
  • Nucleotide Motifs / genetics*
  • Time Factors
  • Transcription, Genetic*
  • Transcriptional Activation / genetics

Substances

  • CRISPR-Associated Protein 9
  • Endonucleases