Cas12a/Guide RNA-Based Platform for Rapid and Accurate Identification of Major Mycobacterium Species.

Mycobacterium tuberculosis infection and nontuberculous mycobacteria (NTM) infections exhibit similar clinical symptoms; however, the therapies for these two types of infections are different. Therefore, the rapid and accurate identification of M. tuberculosis and NTM species is very important for the control of tuberculosis and NTM infections. In the present study, a Cas12a/guide RNA (gRNA)-based platform was developed to identify M. tuberculosis and most NTM species. By designing species-specific gRNA probes targeting the rpoB sequence, a Cas12a/gRNA-based platform successfully identified M. tuberculosis and six major NTM species (Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium gordonae, and Mycobacterium fortuitum) without cross-reactivity. In a blind assessment, a total of 72 out of 73 clinical Mycobacterium isolates were correctly identified, which is consistent with previous rpoB sequencing results. These results suggest that the Cas12a/gRNA-based platform is a promising tool for the rapid, accurate, and cost-effective identification of both M. tuberculosis and NTM species.

Identification of Mycobacterium abscessus species and subspecies using the Cas12a/sgRNA-based nucleic acid detection platform.

The rapidly growing mycobacterium Mycobacterium abscessus is a clinically important organism causing pulmonary and skin diseases. The M. abscessus complex is comprised of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Here, we aimed to develop a Cas12a/sgRNA-based nucleic acid detection platform to identify M. abscessus species and subspecies. By designing specific sgRNA probes targeting rpoB and erm(41), we demonstrated that M. abscessus could be differentiated from other major mycobacterial species and identified at the subspecies level. Using this platform, a total of 38 clinical M. abscessus isolates were identified, 18 as M. abscessus subsp. abscessus and 20 as M. abscessus subsp. massiliense. We concluded that the Cas12a/sgRNA-based nucleic acid detection platform provides an easy-to-use, quick, and cost-effective approach for identification of M. abscessus species and subspecies.

Determination of the predictive factors for diagnostic positivity of nucleic acid amplification tests for diagnosing pulmonary tuberculosis.

Background: Tuberculosis (TB) remains a major threat to human health, and TB diagnostic methods remain unsatisfactory. Nucleic acid amplification tests (NAATs) show higher sensitivity compared with culture for the diagnosis of pulmonary TB (PTB). However, NAATs are expensive and cannot be easily implemented outside major medical centers. To improve the sensitivity of NAATs for PTB diagnosis, we investigated the predictive factors that might optimize NAAT utilization. Methods: A total of 1263 patients with suspected PTB were enrolled for evaluation. The sensitivity, specificity, and accuracy of methods including smear-microbiology, culture of Mtb and NAAT for Mycobacterium tuberculosis (Mtb) detection in sputum and bronchoalveolar lavage fluid samples were compared. Odds ratios and 95% confidence intervals were used to assess variables that might be associated with positive NAAT results for sputum and bronchoalveolar lavage fluid from patients with suspected PTB. Results: NAAT showed higher sensitivity for Mtb detection (61.1%) when compared with smear (9.0%) and Mtb culture (47.8%). We found that an elevated erythrocyte sedimentation rate, the presence of cavities, and positive interferon-γ release assay (IGRA) results were indicative of positive Mtb detection by NAAT. Moreover, individuals who had all three of these characteristics showed an 86% diagnostic positivity for PTB from Mtb detection by NAAT. Conclusions: Our study suggests that an elevated erythrocyte sedimentation rate, a positive IGRA result, and the presence of pulmonary cavities are helpful factors for predicting positive Mtb detection by NAAT. Patients with the three positive clinical markers should undergo NAAT for Mtb detection because they are the most likely individuals to be bacteriologically confirmed as having TB.

CRISPR/Cas12a-based biosensing platform for precise and efficient screening of CRISPR/Cas9-induced biallelic mutants.

CRISPR/Cas9 is a robust tool to manipulate genes in a wide range of species. Although several methods are introduced to identify the CRISPR/Cas9-induced mutations, they are labor-intensive, costly, and not easy to use or were sequence-limited. Moreover, few of them could identify the biallelic mutants that are the desired outcomes of targeted mutagenesis. Recently, a CRISPR/Cas12a-mediated biosensing platform was developed to detect nucleic acids based on the collateral DNA cleavage activity of Cas12a; it was highly sensitive, specific, rapid, and cost-efficient for genotyping, mutation detection, and single nucleotide polymorphism (SNP) identification, thereby deeming it as an innovative method for screening the CRISPR/Cas9-induced biallelic mutants. Thus, the CRISPR/Cas12a-based biosensing platform has been successfully utilized for screening 23 CRISPR/Cas9-induced biallelic mutants in Thp-1 cells, which were also confirmed by direct sequencing and ELISA. The precision and efficiency of CRISPR/Cas12a-based biosensing platform make it a promising tool for screening of CRISPR/Cas9-induced biallelic mutants in the future.