Determinants for Efficient Editing with Cas9-Mediated Recombineering in Escherichia coli.
ACS Synth Biol
; 9(5): 1083-1099, 2020 05 15.
Article
em En
| MEDLINE
| ID: mdl-32298586
ABSTRACT
In E. coli, editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Escherichia coli
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Sistemas CRISPR-Cas
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Edição de Genes
Idioma:
En
Revista:
ACS Synth Biol
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos
País de publicação:
EEUU
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ESTADOS UNIDOS
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ESTADOS UNIDOS DA AMERICA
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EUA
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UNITED STATES
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UNITED STATES OF AMERICA
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US
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USA