Supercas: A Supercharged CRISPR-Cas12a Enzyme for Enhanced Nucleic Acid Detection and Gene Editing

In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized genetic research, offering unparalleled ease and accuracy in gene editing and nucleic acid detection. Particularly, the CRISPR-Cas12a enzyme has shown significant promise in these domains. This paper introduces "SupErCas", a novel variant of the CRISPR-Cas12a ortholog derived from Eubacterium Rectale (ErCas12a) that has been biochemically engineered to possess increased overall charge. We designed and characterized a library of ErCas12a enzymes with increasing positive and negative charges. We used structure-guided mutagenesis to systematically substitute neutral amino acids with those containing either positively charged (Lys, Arg, His) or negatively charged (Glu, Asp) side chains, thereby enriching the local charge at different domains of the enzyme. We observed that increasing the overall charge of the enzyme lead to an increase in its resistance to pH and temperature changes. Our charged Cas12a variants work at a broader temperature and pH range compared to the wild type Cas12a, thus making them ideal candidates for diverse genomic applications. We also demonstrate that one of the negatively charged variants characterized here possesses superior trans-cleavage activity that can be applied for highly sensitive detection of nucleic acid substrates. In summary, our work here explores the effect of charge engineering the CRSPR-Cas12a enzymes and turning these charged variants into tools for a broad range of gene-therapy and diagnostic applications.