Synthetic mismatches enable specific CRISPR-Cas12a-based detection of genome-wide SNVs tracked by ARTEMIS
Kavish A.V. Kohabir, Jasper Linthorst, Lars O. Nooi, Rick Brouwer, Rob M.F. Wolthuis, Erik A. Sistermans
Abstract
<h2>Summary</h2> Detection of pathogenic DNA variants is vital in cancer diagnostics and treatment monitoring. While CRISPR-based diagnostics (CRISPRdx) offer promising avenues for cost-effective, rapid, and point-of-care testing, achieving single-nucleotide detection fidelity remains challenging. We present an <i>in silico</i> pipeline that scans the human genome for targeting pathogenic mutations in the seed region (ARTEMIS), the most stringent crRNA domain. ARTEMIS identified 12% of pathogenic SNVs as Cas12a recognizable, including 928 cancer-associated variants such as <i>BRAF</i><sup>V600E</sup>, <i>BRCA2</i><sup>E1953∗</sup>, <i>TP53</i><sup>V272M</sup>, and <i>ALDH2</i><sup>E504K</sup>. Cas12a exhibited remarkable tolerance to single mismatches within the seed region. Introducing deliberate synthetic mismatches within the seed region yielded on-target activity with single-nucleotide fidelity. Both positioning and nucleobase types of mismatches influenced detection accuracy. With improved specificity, Cas12a could accurately detect and semi-quantify <i>BRAF</i><sup>V600E</sup> in cfDNA from cell lines and patient liquid biopsies. These results provide insights toward rationalized crRNA design for high-fidelity CRISPRdx, supporting personalized and cost-efficient healthcare solutions in oncologic diagnostics.