(27s) Adaptability of 22 Functional Cas12a Orthologs Using a Combinatorial Approach for Nucleic Acid Detection in Clinical Samples

CRISPR-Cas systems are a valuable tool for detecting SARS-CoV-2 among other infectious diseases in addition to applications in human health through genetic engineering. Methods using this system have led to the development of rapid, sensitive, and cost-effective diagnostic methods such as DETECTR and SHERLOCK, which employ Class 2 type V and VI single effector Cas proteins, including Cas12a and Cas13a. These Cas proteins have strong trans-cleavage activity, making them ideal for detecting SARS-CoV-2. In this study, we aimed to improve the detection capabilities of CRISPR-Cas12a systems by exploring the trans-cleavage activity of 22 functional Cas12a variants. Through this investigation, we discovered that Cas12a variants exhibited flexible adaptability towards different non-canonical crRNAs among these orthologs. Notably, unique types of recombination of crRNA significantly enhanced the trans-cleavage activity of some Cas12a variants, where we showed that BoCas12a, BsCas12a, and TsCas12a showed robust trans-cleavage activity through the recombination of specific non-canonical crRNA-Cas12a complexes. Furthermore, we conducted a Differential Scanning Fluorimetry (DSF) assay where crRNA corresponding to Pb2Cas12a showed enhanced thermal stability towards combinations with several other Cas12a proteins in the form of binary complex. We also investigated the tolerance of multiple Cas12a orthologs towards crRNA guides split at different points in the hairpin region as well as split crRNA designs with short ssDNA and RNA extensions stemming from the split hairpin region. This study found that multiple orthologs allow for the recruitment of split crRNA components, including AsCas12a, ErCas12a, and HkCas12a. Out of the variants that were able to tolerate a split crRNA, activity was conserved in multiple orthologs when tested with extended split crRNA components.