High concentration of Cas12a effector tolerates more mismatches on ssDNA
Haibo Li, Xiaojing Cui, Lingxiao Sun, Xiaoyan Deng, Shuai Liu, Xiaohui Zou, Binbin Li, Chunlei Wang, Yeming Wang, Yinmei Liu, Binghuai Lu, Bin Cao
Abstract
Rapid pathogen detection is critical for prompt treatment, interrupting transmission routes, and decreasing morbidity and mortality. The V-type CRISPR system had been used for rapid pathogen detection. However, whether single-stranded DNA in CRISPR system can cause false positives remains undetermined. Herein, we show that high molar concentration of Cas12a effector tolerated more mismatches on ssDNA and activated its trans-cleavage activity at six base matches. Reducing Cas12a and crRNA molar concentration increased the minimal base-match number required for Cas12a ssDNA activation to 11, which reducing nonspecific activation. We then established a Cas12a-based M tuberculosis detection system with a primer having an 8 bp overlap with crRNA. This system did not exhibit primer-induced false positives, and minimum detection copy reached 1 copy/uL (inputting 1-μL sample) in standard strains. The Cas12a-based M tuberculosis detection system showed 80.0% sensitivity and 100.0% specificity in verification using clinical specimens, compared with Xpert MTB/RIF, which showed 72.0% sensitivity and 90.9% specificity. All these results prove that appropriate concentration of cas12a effector can effectively perform nucleic acid detection.