Engineering Highly Thermostable Cas12b via De Novo Structural Analyses for One-Pot Detection of Nucleic Acids

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Abstract Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostics have elevated nucleic acid detection in terms of sensitivity, specificity, and rapidity in recent years. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify workflow and reduce carryover contamination. Here, we report an engineered Cas12b system from Brevibacillus (eBrCas12b) with improved thermostability that falls within the optimal range (60-65°C) of the Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP). Using de novo structural analyses via DeepDDG and HotSpot Wizard based on Alpha Fold and SWISS-MODEL predicted structures, mutations were introduced into the REC and RuvC domains of wild-type BrCas12b to tighten the hydrophobic cores of the protein, thereby enhancing its stability at high temperatures. We expressed, purified, and systematically characterized 49 BrCas12b variants with an emphasis on functionality and thermostability. The assay utilizing eBrCas12b, which we coined SPLENDID ( S ingle- p ot L AMP-mediated e ngineered BrCas12b for n ucleic acid d etection of i nfectious d iseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting—4°C and 7°C higher than wild-type BrCas12b and AapCas12b, respectively. We further validated SPLENDID clinically in 40 Hepatitis C (HCV) positive and 40 negative serum samples. A specificity of 97.5%, an accuracy of 90.0%, and a sensitivity of 82.5% were achieved. Results can be obtained via one-pot testing in as little as 20 minutes. With the extraction process, the entire assay can be performed in under an hour. Therefore, we believe that SPLENDID has the potential to become a widely universal platform for the detection of infectious diseases.

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