The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins constitute the innate adaptive immune system in several bacteria and archaea. This immune system helps them in resisting the invasion of phages and foreign DNA by providing sequence-specific acquired immunity. Owing to the numerous advantages such as ease of use, low cost, high efficiency, good accuracy, and a diverse range of applications, the CRISPR-Cas system has become the most widely used genome editing technology. Hence, the advent of the CRISPR/Cas technology highlights a tremendous potential in clinical diagnosis and could become a powerful asset for modern medicine. This study reviews the recently reported application platforms for screening, diagnosis, and treatment of different diseases based on CRISPR/Cas systems. The limitations, current challenges, and future prospectus are summarized; this article would be a valuable reference for future genome-editing practices.
Object: Mycobacterium tuberculosis (MTB) is a bacterium that can cause zoonoses by aerosol transmission. Tuberculosis (TB) caused by MTB places a heavy burden on world public health security. The development of efficient, specific, convenient, and inexpensive MTB assays is important for the prevention and control of TB. Methods: Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) is a special DNA repeat family widely present in bacterial and archaeal genomes. In this study, we established a specific detection method for MTB using the CRISPR system, combined with recombinase mediated isothermal nucleic acid amplification (RAA) to improve the sensitivity of the detection system and achieve "two-level" amplification of the detection signal. The sensitivity and specificity of RAA combined with the CRISPR/Cas system were analyzed. Using BACTEC 960 culture as the gold standard for the detection of MTB, we established the TB-CRISPR technique by testing 504 samples from patients with suspected tuberculosis. Results: MTB H37Ra could be detected as low as 3.13 CFU/mL by the CRISPR-Cas12a system targeting IS6110. With BACTCE960 culture (120 positives and 384 negatives) as the gold standard, the sensitivity of the TB-CRISPR technique was 0.883 (0.809-0.932) and the specificity was 0.940 (0.910-0.961). According to the receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) reached 0.944 (0.914-0.975) within 95% CI. The positive likelihood ratio (PLR) was 14.747 (9.870-22.035) and the negative likelihood ratio (NLR) was 0.124 (0.076-0.203). The positive predictive value (PPV) was 0.822 (0.742-0.881) and the negative predictive value (NPV) was 0.963 (0.937-0.979). Conclusion: TB-CRISPR realizes the rapid screening and diagnosis of MTB. The whole detection time is less than 1.5h. It is easy to operate and does not need to rely on complex instruments. It is of great significance for the rapid detection of MTB and the clinical diagnosis of TB.
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