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Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

View ORCID ProfileHJ. Benns, View ORCID ProfileM. Storch, View ORCID ProfileJ. Falco, FR. Fisher, E. Alves, View ORCID ProfileCJ. Wincott, View ORCID ProfileJ. Baum, GS. Baldwin, View ORCID ProfileE. Weerapana, View ORCID ProfileEW. Tate, View ORCID ProfileMA. Child
doi: https://doi.org/10.1101/2021.02.04.429737
HJ. Benns
1Department of Life Sciences, Imperial College London, London, UK
4Department of Chemistry, Imperial College London, London, UK
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M. Storch
2London Biofoundry, Imperial College Translation & Innovation Hub, London, UK
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J. Falco
3Department of Chemistry, Boston College, Massachusetts, USA
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FR. Fisher
1Department of Life Sciences, Imperial College London, London, UK
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E. Alves
1Department of Life Sciences, Imperial College London, London, UK
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CJ. Wincott
1Department of Life Sciences, Imperial College London, London, UK
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J. Baum
1Department of Life Sciences, Imperial College London, London, UK
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GS. Baldwin
1Department of Life Sciences, Imperial College London, London, UK
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E. Weerapana
3Department of Chemistry, Boston College, Massachusetts, USA
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EW. Tate
4Department of Chemistry, Imperial College London, London, UK
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  • For correspondence: m.child@imperial.ac.uk
MA. Child
1Department of Life Sciences, Imperial College London, London, UK
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  • For correspondence: m.child@imperial.ac.uk
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Summary

Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of >1,000 cysteines on ~700 proteins in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed >800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted February 04, 2021.
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Prioritization of antimicrobial targets by CRISPR-based oligo recombineering
HJ. Benns, M. Storch, J. Falco, FR. Fisher, E. Alves, CJ. Wincott, J. Baum, GS. Baldwin, E. Weerapana, EW. Tate, MA. Child
bioRxiv 2021.02.04.429737; doi: https://doi.org/10.1101/2021.02.04.429737
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Prioritization of antimicrobial targets by CRISPR-based oligo recombineering
HJ. Benns, M. Storch, J. Falco, FR. Fisher, E. Alves, CJ. Wincott, J. Baum, GS. Baldwin, E. Weerapana, EW. Tate, MA. Child
bioRxiv 2021.02.04.429737; doi: https://doi.org/10.1101/2021.02.04.429737

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