Point-of-care DNA diagnostics from raw samples

Project Summary The proposed project will develop living biosensors for detecting and analyzing DNA at the single- base level, without requiring sample purification or any equipment. DNA is the prime information carrier for life, and DNA analysis provides valuable information for, e.g., diagnosing microbial infections or tracking disease outbreaks. Many techniques exist for detecting and analyzing DNA, but these generally require processing steps to extract and purify samples, and most require expensive equipment and significant training and expertise. This proposal will transfer that complexity into the biosensor itself, harnessing functions that evolved into living bacteria over billions of years to pull DNA out of raw samples, analyze it, and produce easily read output. The biosensors will pull in DNA using natural competence, and analyze it with single-base precision using their endogenous CRISPR-Cas system. Upon detecting a target sequence, the living biosensors will release thousands of signal molecules that can be detected using a lateral flow assay, similar to a consumer pregnancy or Covid-19 test. Several target DNA sequences will be used for demonstrations: urinary tract pathogens, E. coli, and Salmonella. The target uropathogens are difficult to diagnose with standard culture tests. Using single-base sequence analysis, the biosensors will subtype E. coli as likely pathogenic or likely commensal. A similar strategy will be employed to detect single-base mutations responsible for the majority of fluoroquinolone-resistant Salmonella isolates. DNA biosensing will be demonstrated in clinically relevant human samples, without the extensive purification required by other methods. The result will be a hybrid living biosensor / lateral flow assay that requires minimal sample preparation, produces rapid results, and can achieve single-base resolution. The biosensors developed in this project could find applications any time DNA monitoring is needed that is inexpensive, requires minimal sample preparation, equipment, and expertise, or takes place at the point of care. Examples include clinical diagnostics, monitoring disease outbreaks for public health, or environmental monitoring, with particular benefits where resources are limited.