CRISPR /Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli
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.
- Authors:
-
[5]
- Department of Chemical and Biological Engineering University of Colorado Boulder CO USA, IAME INSERM Université de Paris Paris France
- Department of Chemical and Biological Engineering University of Colorado Boulder CO USA
- Renewable &, Sustainable Energy Institute University of Colorado Boulder CO USA
- IAME INSERM Université de Paris Paris France
- Department of Chemical and Biological Engineering University of Colorado Boulder CO USA, Renewable &, Sustainable Energy Institute University of Colorado Boulder CO USA, Novo Nordisk Foundation Center for Biosustainability Danish Technical University Copenhagen Denmark
- Publication Date:
- Research Org.:
- Univ. of Colorado, Boulder, CO (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); European Research Council (ERC)
- OSTI Identifier:
- 1604825
- Alternate Identifier(s):
- OSTI ID: 1604826; OSTI ID: 1617013
- Grant/Contract Number:
- SC0018368
- Resource Type:
- Published Article
- Journal Name:
- Molecular Systems Biology
- Additional Journal Information:
- Journal Name: Molecular Systems Biology Journal Volume: 16 Journal Issue: 3; Journal ID: ISSN 1744-4292
- Publisher:
- Wiley
- Country of Publication:
- United Kingdom
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; CRISPR-Cas9; deep mutational scanning; essential genes; genome diting; recombineering
Citation Formats
Choudhury, Alaksh, Fenster, Jacob A., Fankhauser, Reilly G., Kaar, Joel L., Tenaillon, Olivier, and Gill, Ryan T. CRISPR /Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli. United Kingdom: N. p., 2020.
Web. doi:10.15252/msb.20199265.
Choudhury, Alaksh, Fenster, Jacob A., Fankhauser, Reilly G., Kaar, Joel L., Tenaillon, Olivier, & Gill, Ryan T. CRISPR /Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli. United Kingdom. https://doi.org/10.15252/msb.20199265
Choudhury, Alaksh, Fenster, Jacob A., Fankhauser, Reilly G., Kaar, Joel L., Tenaillon, Olivier, and Gill, Ryan T. Sun .
"CRISPR /Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli". United Kingdom. https://doi.org/10.15252/msb.20199265.
@article{osti_1604825,
title = {CRISPR /Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli},
author = {Choudhury, Alaksh and Fenster, Jacob A. and Fankhauser, Reilly G. and Kaar, Joel L. and Tenaillon, Olivier and Gill, Ryan T.},
abstractNote = {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.},
doi = {10.15252/msb.20199265},
journal = {Molecular Systems Biology},
number = 3,
volume = 16,
place = {United Kingdom},
year = {Sun Mar 01 00:00:00 EST 2020},
month = {Sun Mar 01 00:00:00 EST 2020}
}
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