CRISPR /Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli

Choudhury, Alaksh; Fenster, Jacob A.; Fankhauser, Reilly G.; Kaar, Joel L.; Tenaillon, Olivier; Gill, Ryan T.

doi:10.15252/msb.20199265

Title: CRISPR /Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli

Journal Article · 16 March 2020 · Molecular Systems Biology

DOI: https://doi.org/10.15252/msb.20199265 · OSTI ID:1604825

Choudhury, Alaksh ^[1]; Fenster, Jacob A. ^[2]; Fankhauser, Reilly G. ^[2]; Kaar, Joel L. ^[2]; Tenaillon, Olivier ^[3];

^[4]

Univ. of Colorado, Boulder, CO (United States); Univ. of Paris (France). Infection Antimicrobials Modelling Evolution (IAME) and French National Inst. of Health and Medical Research (INSERM)
Univ. of Colorado, Boulder, CO (United States)
Univ. of Paris (France). Infection Antimicrobials Modelling Evolution (IAME) and French National Inst. of Health and Medical Research (INSERM)
Univ. of Colorado, Boulder, CO (United States); Danish Technical Univ., Copenhagen (Denmark)

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.

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Research Organization:: Univ. of Colorado, Boulder, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); European Research Council (ERC)

Grant/Contract Number:: SC0018368

OSTI ID:: 1604825

Alternate ID(s):: OSTI ID: 1604826; OSTI ID: 1617013

Journal Information:: Molecular Systems Biology, Vol. 16, Issue 3; ISSN 1744-4292

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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