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Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9
Gigi C. G. Choi1; Peng Zhou1; Chaya T. L. Yuen1; Becky K. C. Chan1; Feng Xu1; Siyu Bao2; Hoi Yee Chu2; Dawn Thean1; Kaeling Tan3,4; Koon Ho Wong3,5; Zongli Zheng2,6,7; Alan S. L. Wong1,8
2019-08
Source PublicationNATURE METHODS
ISSN1548-7091
Volume16Issue:8Pages:722–730
AbstractThe 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.
DOI10.1038/s41592-019-0473-0
Indexed BySCI
Language英语
WOS Research AreaBiochemistry & Molecular Biology
WOS SubjectBiochemical Research Methods
WOS IDWOS:000477857700028
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Document TypeJournal article
CollectionFaculty of Health Sciences
Affiliation1.Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China
2.Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong, China
3.Faculty of Health Sciences, University of Macau, Macau, China
4.Genomics, Bioinformatics and Single Cell Analysis Core, Faculty of Health Sciences, University of Macau, Macau, China
5.Institute of Translational Medicine, University of Macau, Macau, China
6.Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China
7.Biotechnology and Health Centre, City University of Hong Kong Shenzhen Research Institute, Shenzhen, China
8.Department of Electrical and Electronic Engineering, The University of Hong Kong, Hong Kong, China
Recommended Citation
GB/T 7714
Gigi C. G. Choi,Peng Zhou,Chaya T. L. Yuen,et al. Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9[J]. NATURE METHODS,2019,16(8):722–730.
APA Gigi C. G. Choi.,Peng Zhou.,Chaya T. L. Yuen.,Becky K. C. Chan.,Feng Xu.,...&Alan S. L. Wong.(2019).Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.NATURE METHODS,16(8),722–730.
MLA Gigi C. G. Choi,et al."Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9".NATURE METHODS 16.8(2019):722–730.
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