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Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.

Gigi C G ChoiPeng ZhouChaya T L YuenBecky K C ChanFeng XuSiyu BaoHoi Yee ChuDawn TheanKaeling TanKoon Ho WongZongli ZhengAlan S L Wong
Published in: Nature methods (2019)
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.
Keyphrases
  • crispr cas
  • genome editing
  • high throughput
  • copy number
  • protein protein
  • amino acid
  • binding protein
  • escherichia coli
  • small molecule
  • single cell
  • genome wide