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Inactive Cas13 orthologs have been fused to a mutant human ADAR2 deaminase domain at the C terminus to enable programmable adenosine-to-inosine (A-to-I) RNA editing in selected transcripts. Although promising, existing RNA-editing tools generally suffer from a trade-off between efficacy and specificity, and off-target editing remains an unsolved problem. Here we describe the development of an optimized RNA-editing platform by rational protein engineering, CasRx-based Programmable Editing of RNA Technology (xPERT). We demonstrate that the topological rearrangement of a CasRx K940L mutant by circular permutation results in a robust scaffold for the tethering of a deaminase domain. We benchmark our tool against the REPAIR system and show that xPERT exhibits strong on-target activity like REPAIRv1 but low off-target editing like REPAIRv2. Our xPERT platform can be used to alter RNA sequence information without risking genome damage, effect temporary cellular changes and customize protein function., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.