Engineered FnCas12a with enhanced activity through directional evolution in human cells.
J Biol Chem
; 296: 100394, 2021.
Article
em En
| MEDLINE
| ID: mdl-33567342
ABSTRACT
Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Retinosquise
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Endodesoxirribonucleases
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Proteínas do Olho
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Proteínas Associadas a CRISPR
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Francisella
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Mutação
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article