CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli.
Mol Syst Biol
; 16(3): e9265, 2020 03.
Article
en En
| MEDLINE
| ID: mdl-32175691
ABSTRACT
Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Ingeniería Genética
/
Genes Esenciales
/
Escherichia coli
/
Mutación
Idioma:
En
Revista:
Mol Syst Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOTECNOLOGIA
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos