Sequence to activity relationship (SAR) mapping is essential to almost all efforts seeking to identify biological mechanisms1?10. Even though considerable advances have occurred, SAR mapping methods remain slow, laborious, and limited to scales that are not readily extended beyond a single loci. Such limitations are significant in the context of target identification, small molecule screening, protein therapeutics, therapeutic production, and antimicrobial resistance, among others. We have spent the last decade focused on the development of SAR mapping technologies that address these limitations5,6,11. Most recently we have developed a breakthrough technology (Garst et al, 2016, Nature Biotechnology In Press 33) that combines multiplex DNA oligomer synthesis, phage-based recombineering, and the CRISPR/Cas9 system to enable SAR mapping at a scale orders of magnitude beyond prior approaches. Here, we will expand this approach to demonstrate SAR mapping at a new scale (pathway level SAR mapping) and in a clinically relevant model system. We specifically propose to comprehensively map sequence to activity across the DXP pathway; an essential pathway in a range of pathogens including various bacteria and apicomplexan protozoa such as the malaria-causing Plasmodium falciparum (Pf). While treatment with inhibitors of this pathway can be effective12,13, resistance eventually emerges. Sequence-activity mapping of this pathway in the presence and absence of specific inhibitors will not only identify a range of targets for inhibiting this pathway but also uncover mutations leading to resistance. Thus, our efforts here will result in new specific understanding about a clinically important pathway as well as more broadly demonstrate a new platform for SAR mapping that extends to the pathway scale. At the end of this R21 program, we will specifically have generated quantitative data relating each of several tens of thousands targeted sequence alterations to the activity of the essential DXP pathway. The maps will be generated not only for the native Ec pathway but also for the clinically relevant pathway taken from the malaria pathogen P. falciparum. All methods and data will be broadly disseminated to aide in the adoption of this new approach for multiplex SAR mapping. Importantly, this proposal aligns well with the NIAID research agenda for malaria, specifically to better understand and confront drug resistance and elucidate mechanisms of drug resistance14.
Sequence to activity relationship (SAR) mapping is essential to almost all efforts seeking to identify biological mechanisms1?10, including a broad range of studies focused on combating infectious diseases. Here, we will demonstrate a breakthrough SAR mapping technology that combines multiplex DNA synthesis, high- throughput genome modification methods, and CRISPR/Cas9 based editing in the context of antimicrobial drug development and resistance. In particular, we will develop SAR maps for the entire DXP pathway (50,000+ designer mutations), which is a target of anti-malarial drug development programs of interest to NIH (NIAID) and more broadly infectious disease research in general.
