Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9

Nat Methods. 2019 Aug;16(8):722-730. doi: 10.1038/s41592-019-0473-0. Epub 2019 Jul 15.

Abstract

The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Cas Systems / genetics*
  • Gene Editing*
  • Humans
  • Mutagenesis*
  • Mutation*
  • Protein Engineering
  • RNA, Guide, CRISPR-Cas Systems
  • Software*
  • Streptococcus pyogenes / enzymology
  • Substrate Specificity

Substances

  • CRISPR-Associated Protein 9