Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13

Abstract

Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets. We demonstrate that this strategy of tiling improves cfDNA detection compared to amplification and detection of a single targeted locus. WATSON can detect cfDNA from Mycobacterium tuberculosis in plasma of patients with active pulmonary tuberculosis, a disease that urgently needs accurate, minimally-invasive, field-deployable diagnostics. We thus demonstrate the potential for translating WATSON to a lateral flow platform. WATSON demonstrates the ability to capitalize on the strengths of targeting microbial cfDNA to address the need for point-of-care diagnostic tests for infectious diseases.

Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies

“…Furthermore, given that our priming agents modulate cfDNA clearance, their use could be considered in applications beyond oncology. Priming could improve detection of microbial cfDNA during early or deep-seated infections ( 70 ), where diagnosis is critical for therapy selection but remains challenging. Liquid biopsy–based applications in cardiovascular disease and Alzheimer’s disease are other areas where the low abundance of cfDNA is a limitation, and where priming agents may be beneficial ( 71 , 72 ).…”

A new method for the detection of Mycobacterium tuberculosis based on the CRISPR/Cas system

“…Four gRNAs designed for IS6110 and IS1081 were compared longitudinally based on fluorescence profiles. Under the same concentration of the bacterial solution, H37Ra contains 17 copies of IS6110 and only 5–7 copies of IS1081 [ 17 , 29 ]. Therefore, IS6110 reached the fluorescence platform faster than IS1081 after RAA isothermal amplification and could detect as low as 53 copies /mL, indicating that CRISPR/Cas12a is an ultra-sensitive detection technology.…”

Outlook for CRISPR-based tuberculosis assays now in their infancy

“…Most reported assays still adhere to the original design paradigm of CRISPR assays, where CRISPR-based signal amplification is performed after an exponential nucleic acid amplification (NAA) step (21,44,45). This assay design detects trace levels of target NA sequences in clinical specimens to yield high analytical sensitivity (30,34,36,41), but can prolong run times (> 1 h) (32,33,36,39,40,43) (Figure 1A) and increase the risk of cross-contamination if amplified target NA sequences are transferred to separate CRISPR reactions. Most of these CRISPR assays employ recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP)-based isothermal amplification reactions for NAA to avoid the need for a thermocycler, which can facilitate the development of POC applications.…”

“…Most of these CRISPR assays employ recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP)-based isothermal amplification reactions for NAA to avoid the need for a thermocycler, which can facilitate the development of POC applications. However, many of these assays also generate fluorescent signals that require additional equipment to read and are thus less suitable for use in remote and resource-limited areas without the development of simple readout devices, although lateral flow strip-based visual readout approaches can present a good alternative for qualitative assays (37,41,43).…”

“…Several groups have realized the potential of CRISPR-based assays to overcome weaknesses associated with current tests employed for TB diagnosis (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). However, their studies largely ignore the drawbacks and challenges of CRISPR-based TB (CRISPR-TB) assays for methodology development and clinical validation studies, as these applications are still in their infancy.…”