PROJECT SUMMARY Low-resource settings have a high burden of Neisseria gonorrhoeae infection, but insufficient laboratory infrastructure for diagnostic tests and antimicrobial susceptibility determination, leading to syndromic management, which misses a high proportion of cases and results in the overuse of antibiotics. Together, those factors contribute to driving the emergence of antimicrobial resistance (AMR), a global public health threat. Two novel strategies can combine to address those challenges. First, the use of CRISPR-based technology permits the development of low-cost, point-of-care (POC) assays, which can be used in resource-limited settings. Second, determination of molecular resistance markers can facilitate resistance-guided therapy, thereby reducing the selective pressure towards AMR. The study aims are: 1) adapt and validate existing CRISPR-based assays for POC detection of N. gonorrhoeae and ciprofloxacin susceptibility, 2) develop a multiplexed CRISPR-based assay for N. gonorrhoeae cefixime susceptibility, and 3) evaluate the feasibility and acceptability of the POC N. gonorrhoeae and AMR assays among two health centers in Botswana. The study is based upon our preliminary work in the development of Cas13a-based diagnostics for N. gonorrhoeae and ciprofloxacin susceptibility, and will leverage clinical specimens collected as a part of an ongoing trial at the Massachusetts General Hospital Sexual Health Clinic, as well as through existing collaborations in Botswana. This Mentored Patient-Oriented Research Career Development Award (K23) supports the career development of Dr. Lao-Tzu Allan-Blitz, an instructor of medicine with experience in the epidemiology of and novel diagnostic assays targeting AMR sexually transmitted infections (STI). The K23 will support Dr. Allan-Blitz to develop expertise in 1) assay development for POC detection, and 2) assay implementation and evaluation. To achieve the proposed research and training aims, Dr. Allan-Blitz has assembled a team of mentors who are world experts. Dr. Jacob Lemieux, who is an expert in metagenomics, DNA sequencing, and CRISPR-based assay development, will serve as the primary mentor. In addition, his Co-Mentorship team includes Dr. Pardis Sabeti, a world expert in computational genetics, microbial genomics, diagnostic technologies, and disease surveillance in Africa, and Dr. Shahin Lockman, a world leader in implementation science research with longstanding collaborations in Botswana. The proposed research and training aims will leverage strong, longstanding collaborations and the robust research infrastructure between Brigham and Women's Hospital, Massachusetts General Hospital, the Broad Institute, and the Botswana Harvard AIDS Institute Partnership. Finally, the K23 award will advance Dr. Allan-Blitz's career goal of becoming an expert in assay development for AMR STI and assay implementation, as well as facilitate his transition to research independence.