Abstract
Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR. This selection is especially challenging since it is relatively weak in E. coli, with multiple naturally occurring pathways for resistance. We identified several previously unreported mutations that confer fosmidomycin resistance, in highly conserved sites that also exist in pathogens including the malaria-inducing Plasmodium falciparum. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs.
Original language | English |
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Pages (from-to) | 2824-2832 |
Number of pages | 9 |
Journal | ACS Synthetic Biology |
Volume | 7 |
Issue number | 12 |
DOIs | |
State | Published - Nov 21 2018 |
Externally published | Yes |
Funding
We would like to thank Yael David for her helpful insights and Emily Freed for her help with the manuscript preparation. This work was funded both by the US Department of Energy Grant No. DE-SC008812 and by the National Institute of Allergy and Infectious Diseases (NIAID) at the NIH, Grant Number 1R21AI128296-01A1
Keywords
- CRISPR/Cas9
- acquired resistance
- deoxyxylulose phosphate reductoisomerase
- fosmidomycin
- malaria
- sequence to activity mapping