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

Autor: Choi GCG; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China., Zhou P; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China., Yuen CTL; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China., Chan BKC; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China., Xu F; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China., Bao S; Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong, China., Chu HY; Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong, China., Thean D; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China., Tan K; Faculty of Health Sciences, University of Macau, Macau, China.; Genomics, Bioinformatics and Single Cell Analysis Core, Faculty of Health Sciences, University of Macau, Macau, China., Wong KH; Faculty of Health Sciences, University of Macau, Macau, China.; Institute of Translational Medicine, University of Macau, Macau, China., Zheng Z; Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong, China.; Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China.; Biotechnology and Health Centre, City University of Hong Kong Shenzhen Research Institute, Shenzhen, China., Wong ASL; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China. aslw@hku.hk.; Department of Electrical and Electronic Engineering, The University of Hong Kong, Hong Kong, China. aslw@hku.hk.
Jazyk: angličtina
Zdroj: Nature methods [Nat Methods] 2019 Aug; Vol. 16 (8), pp. 722-730. Date of Electronic Publication: 2019 Jul 15.
DOI: 10.1038/s41592-019-0473-0
Abstrakt: 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.
Databáze: MEDLINE
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