CRISPR-Based Diagnostics: A Potential Tool to Address the Diagnostic Challenges of Tuberculosis

Abstract

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, which infects more than 23% of the world’s population. With the emergence of drug-resistant TB (DR-TB) and latent TB infection (LTBI), rapid diagnosis of DR-TB and LTBI has become a challenge for the prevention and control of TB. Herein, we highlight these challenges and discuss emerging clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics in TB detection. Currently, the clinical diagnosis of M. tuberculosis infection mainly depends on pathogenic and molecular biological methods, including sputum smear, sputum culture, and Xpert. Although CRISPR-based diagnostics have not been applied to the clinical diagnosis of TB, they have shown exciting preponderances in TB diagnosis compared with traditional methods, including higher sensitivity, less sample input, and shorter turnaround time. CRISPR-based diagnostics represent a potential tool to address the challenges and natural weaknesses associated with traditional TB diagnosis methods. Based on the currently available data, we suggest that future CRISPR-based TB diagnostics should be developed in the direction of automation, modularization, diversification, and intelligence. By combining the CRISPR platform with various systems, such as microfluidic chips, droplet microfluidics, electrochemical techniques, and optical systems, the specificity and sensitivity of TB diagnosis may be revolutionized.

Marginal notes, November 2022. From crisis to crisis

“…By contrast, CRISPR can detect microbial DNA or RNA at attamolar concentrations, further improving sensitivity. An interesting angle on this approach is to combine nucleic acid amplification with CRISPR for M. tuberculosis detection at very low copy number and high specificity [2]. This is a promising start but there are still important obstacles to putting this into practice, such as development of a single-tube assay and the introduction of multiple gene targets.…”

Advancements in CRISPR-Based Biosensing for Next-Gen Point of Care Diagnostic Application

“…Moreover, aside from SHERLOCK, which was recently approved by the FDA in 2020 for use as an emergency alternative for the diagnosis of SARS-CoV-2 in the USA, most other POCT solutions discussed in the scope of this article have not yet been widely applied in the healthcare system when seen in the light of the use of RT-PCR and similar conventional approaches. Further development and improvement of these CRISPR detection methods remain necessary for broader and greater practical applications in POCT settings [158,159].…”

“…The majority of CRISPR biosensors is focused on the detection of nucleic acid, while the detection of non-nucleic acid faces a major challenge due to the collateral cleavage property of the Cas protein that requires a method to transduce the target recognition, which is activated by the binding activity of crRNA to its DNA, and even though a solution involving binding activities of allosteric transcription factors (TFs) of bacteria to detect dsDNA and small molecules has been developed, it is still limited [159]. Additionally, sample pretreatment is an important aspect when considering nucleic acid testing, which includes food testing and manually performing POC applications; currently, every biosensor based on CRISPR requires pre-treatment of the raw sample to the screen-out virus; to solve this issue, HUDSON was developed that allows for the amplification of the target instantly after heating it, but the sensitivity of detection is lower than methods used for RNA/DNA detection; therefore, in the future, robust Casprotein is required to solve this problem [159]. Another persistent problem with CRISPR is that in the case of in vivo detection using CRISPR, the complex nature of chromosomes and the cellular environment of the cell make CRISPR produce low searching efficiency.…”

The future of CRISPR in Mycobacterium tuberculosis infection

“…Based on similar designs, several experimental tests were evaluated for the diagnosis of tuberculosis, which were recently reviewed elsewhere [ 49 ]. These methods target conserved genes from the M. tuberculosis genome such as the IS6110 and combine isothermal pre-amplification with Cas12 or Cas13-based detection methods.…”