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Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9
release_odj2ycrbw5emvchyafq45patny
by
Gigi C. G. Choi,
Peng Zhou,
Chaya T. L. Yuen,
Becky K. C. Chan,
Feng Xu,
Siyu Bao,
HOI YEE CHU,
Dawn Thean,
Kaeling Tan,
Koon Ho Wong,
Zongli Zheng,
Alan S. L. Wong
Published
in Nature Methods
by Springer Science and Business Media LLC.
2019 Volume 16, Issue 8, p722-730
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.
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