Break

CRISPR-Cas systems have emerged as a widely adopted diagnostic tool for the detection of SARS-CoV-2 within the past few years. Class 2 type V and VI single effector Cas proteins, such as Cas12a and Cas13a, have been employed for the development of rapid, sensitive, and cost-effective detection platforms including DETECTR and SHERLOCK due to their robust trans-cleavage activity. Drawing inspiration from these projects, we sought to improve the detection capabilities of the CRISPR-Cas12a systems by exploring the trans-cleavage activity of 22 functional Cas12a variants. These Cas12a orthologs share a highly homologous crRNA sequence with exception of the loop region between stem left and stem right of the structured crRNA. 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. Through Differential Scanning Fluorimetry (DSF) assay, crRNA corresponding to Pb2Cas12a showed enhanced thermal stability towards several other Cas12a proteins in the form of binary complex. In addition, we showed that BoCas12a, BsCas12a, and TsCas12a showed robust trans-cleavage activity through the recombination of specific non-canonical crRNA-Cas12a complexes. We applied these systems towards the detection of SARS-CoV-2 and breast cancer patient samples with high sensitivity. In addition to enhanced trans-cleavage capabilities, we investigated multiple Cas12a orthologs tolerance of 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. We 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.