[Development and preliminary clinical evaluation of a CRISPR-AaCas12b-based nucleic acid detection method for...

Objective To express and purify Alicyclobacillus acidiphilus Cas12b (AaCas12b) protein, and establish a rapid clustered regularly interspaced short palindromic repeats (CRISPR)-based assay for Mycobacterium tuberculosis (Mtb) detection, thereby providing a novel tool for clinical diagnosis of tuberculosis (TB). Methods Following construction and transformation of the recombinant expression vector into E. coli BL21 (DE3), soluble expression of the recombinant AaCas12b protein was induced with low-concentration isopropyl β-D-thiogalactopyranoside (IPTG) at low temperature, followed by purification using nickel-affinity chromatography. Subsequently, a specific recombinase polymerase amplification (RPA) assay targeting the IS6110 insertion sequence of Mtb was developed, and the corresponding single guide RNA (sgRNA) was prepared via in vitro transcription. Then a novel CRISPR-AaCas12b detection assay for Mtb was established, and its diagnostic performance was systematically evaluated against conventional clinical methods including acid-fast staining, T-SPOT.TB assay, Mycobacterium culture, and GeneXpert assay. Results A prokaryotic expression and purification system for recombinant AaCas12b was successfully established, yielding AaCas12b protein with activity at a concentration of 13.42 mg/mL and a recovery rate of 61.45%. A specific RPA-CRISPR-AaCas12b detection system was established targeting the IS6110 sequence, with a limit of detection (LOD) of 1.5 CFU/mL for Mtb. In clinical sample validation, the system achieved a sensitivity of 95.83% (95% CI: 79.97%~99.27%), a specificity of 92.31% (95% CI: 75.88%~97.88%), and an accuracy of 94.00% (95% CI: 83.78%~97.93%). Conclusion The established AaCas12b protein preparation system is efficient and stable, enabling the production of low-cost, high-activity tool enzymes. The RPA-CRISPR-AaCas12b TB diagnostic assay targeting the IS6110 sequence exhibits excellent specificity and sensitivity, providing a technical reference and experimental basis for the further development of rapid TB diagnostic platforms.