Abstract：
Experimentally, Cas12a can recognize multiple protospacer adjacent motif (PAM) sequences and is not restricted to the \"TTTN\". However, the application of the CRISPR/Cas12a system is still limited by the PAM for double-stranded DNA (dsDNA). Here, we developed asymmetric RPA (Asy-RPA) to completely break the limitations of PAM. Asy-RPA not only achieved efficient amplification but also converted dsDNA to single-stranded DNA (ssDNA) without complicated steps. The ssDNA products activated the trans-cleavage activity of Cas12a, outputting signals. The application of Asy-RPA completely freed Cas12a from the PAM, which can be more widely used in nucleic acid detection, such as lumpy skin disease virus, with an actual detection limit as low as 1.21 × 101 copies·μL-1. More importantly, Cas12a was intolerant to mutations on ssDNA. This provided technical support for the detection and identification of wild-type Mycobacterium tuberculosis (WT-TB) and rifampin-resistant mutant-type M. tuberculosis (MT-TB). The detection limit was as low as 1 fM for 1% mixed samples. The detection and availability of different treatment options for treatment-resistant and WT-TB were significant for the elimination of TB. In summary, the platform consisting of Asy-RPA and CRISPR/Cas12a was suitable for the detection of various viruses and bacteria and was a boon for the detection of dsDNA without recognizable PAM.
摘要：
实验上,Cas12a可以识别多个原型间隔区相邻基序（PAM）序列，并且不限于“TTTN”。然而,CRISPR/Cas12a系统的应用仍然受到用于双链DNA(dsDNA)的PAM的限制。这里,我们开发了非对称RPA（Asy-RPA），以完全打破PAM的局限性。Asy-RPA不仅实现了有效的扩增，而且无需复杂的步骤即可将dsDNA转化为单链DNA（ssDNA）。ssDNA产物激活了Cas12a的反式切割活性，输出信号。Asy-RPA的应用完全从PAM中释放了Cas12a，可以更广泛地用于核酸检测，如块状皮肤病病毒，实际检测限低至1.21×101拷贝·μL-1。更重要的是,Cas12a对ssDNA上的突变不耐受。这为野生型结核分枝杆菌(WT-TB)和利福平抗性突变型结核分枝杆菌(MT-TB)的检测和鉴定提供了技术支持。1%混合样品的检测限低至1fM。用于治疗抗性和WT-TB的不同治疗选择的检测和可用性对于TB的消除是重要的。总之,由Asy-RPA和CRISPR/Cas12a组成的平台适用于检测各种病毒和细菌,并且是检测没有可识别的PAM的dsDNA的福音.
