Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.
Nat Methods
; 16(8): 722-730, 2019 08.
Article
em En
| MEDLINE
| ID: mdl-31308554
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Software
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Mutagênese
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RNA Guia de Cinetoplastídeos
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Sistemas CRISPR-Cas
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Edição de Genes
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Proteína 9 Associada à CRISPR
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Mutação
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article