Existing CRISPR/Cas12a-based diagnostic platforms offer accurate and vigorous monitoring of nucleic acid targets, but have the potential to be further optimized for more efficient detection. Here, we profiled 16 Cas12a orthologs, focusing on their trans-cleavage activity and their potential as diagnostic enzymes. We observed the Mb2Cas12a has more robust trans-cleavage activity than other orthologs, especially at lower temperatures. An engineered Mb2Cas12a-RRVRR variant presented robust trans-cleavage activity and looser PAM constraints. Moreover, we found the existing one-pot assay, which simultaneously performed Recombinase Polymerase Amplification (RPA) and Cas12a reaction in one system, resulted in the loss of single-base discrimination during diagnosis. Therefore, we designed a reaction vessel that physically separated the RPA and Cas12a steps while maintaining a closed system. This isolated but closed system made diagnostics more sensitive and specific and effectively prevented contamination. This shelved Mb2Cas12a-RRVRR variant-mediated assay detected various targets in less than 15 min and exhibited equal or greater sensitivity than qPCR when detecting bacterial pathogens, plant RNA viruses and genetically modified crops. Overall, our findings further improved the efficiency of the current CRISPR-based diagnostic system and undoubtedly have great potential for highly sensitive and specific detection of multiple sample types.

中文翻译：

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工程改造的 CRISPR-Mb2Cas12a 变体能够在现场进行灵敏、快速的基于核酸的病原体诊断

现有的基于 CRISPR/Cas12a 的诊断平台可对核酸靶标进行准确而有力的监测，但有可能进一步优化以实现更有效的检测。在这里，我们分析了 16 个 Cas12a 直系同源物，重点关注它们的反式切割活性及其作为诊断酶的潜力。我们观察到 Mb2Cas12a比其他直系同源物具有更强的反式裂解活性，特别是在较低温度下。工程化的 Mb2Cas12a-RRVRR 变体呈现出强大的反式-裂解活性和更宽松的 PAM 限制。此外，我们发现现有的一锅法在一个系统中同时进行重组酶聚合酶扩增（RPA）和Cas12a反应，导致诊断过程中单碱基辨别力的丧失。因此，我们设计了一个反应容器，在物理上分隔 RPA 和 Cas12a 步骤，同时保持封闭系统。这种隔离但封闭的系统使诊断更加灵敏和具体，并有效防止了污染。这种搁置的 Mb2Cas12a-RRVRR 变异介导的检测可在不到 15 分钟内检测到各种靶标，并且在检测细菌病原体、植物 RNA 病毒和转基因作物时表现出与 qPCR 相同或更高的灵敏度。全面的，