Charge engineering of ErCas12a to reduce temperature and pH sensitivity for diagnostics

Introduction: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems are currently an indispensable tool for gene editing and diagnostic purposes1,2. Despite significant advancements in the field, there is a need for robust Cas enzymes with broader physiological tolerances. Current gene editing and diagnostic CRISPR-Cas systems are severely limited by the temperature3 and pH4 conditions they can withstand, presenting hindrances when pairing with amplification techniques, such as Reverse-Transcription Loop Mediated Isothermal Amplification (RT-LAMP)3, or detecting from pH-sensitive matrices, including blood and urine. In this study, we show that introducing net charges on ErCas12a enhances cis and trans-cleavage activity significantly at varying pH and temperature. By artificially positively charging a Cas enzyme, it is possible the interactions with the guide RNA would be strengthened. Additionally, depending on charge polarity, the enzyme will favor certain conditions, such as lower pHs and higher temperatures, and vice versa, expanding the environmental capabilities of ErCas12a. These new variants, termed SupErCas proteins, show great promise as versatile diagnostic platforms.

Materials and

Methods: 22 Cas12a orthologs were aligned in SnapGene. Using conservation percentages, we identified either conserved positive or negative amino acids, present in at least 50% of the orthologs. After purifying SupErCas protein variants, we evaluated trans-cleavage activity using a FRET-based assay incorporating dsDNA target activator, fluorescent-quencher ssDNA molecules in a range of pH buffers, as well as checking for temperature sensitivity. Similarly, cis-cleavage activity was verified via gel electrophoresis for three pH and six temperature conditions. Finally, SupErCas proteins were compared to an advanced CRISPR diagnostic test, ENHANCE5, as well implemented in a SAHARA6 assay, used for RNA detection.

Results, Conclusions, and Discussions: Using a rational charge engineered design, we were able to show robust activity at pH and temperature conditions previously undetectable by WT ErCas12a. The negatively charged variant was able to enhance activity at higher temperatures, as well as higher pHs. Both variants displayed a greater tolerance at high pH, where WT had little to no activity. For cis-cleavage, optimal temperature was shifted from 37º C to 42º C. Surprisingly, SupErNeg significantly increased detection capabilities for 500 pM synthetic malaria detection compared to Enhancev1 and v25, allowing for faster and more sensitive diagnosis. Compared to WT, both SupErPos and SupErNeg exhibited much stronger fluorescence detecting RNA at pH 7, exhibiting their versatility for RNA and DNA detection.By engineering conserved residues, we extended the pH and temperature range of Cas proteins while maintaining or enhancing catalytic activity. This approach addresses the need for enzymes that function in non-physiological conditions and accelerates the characterization of novel Cas enzymes in the CRISPR-Cas field.

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