A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18.

ABSTRACT: CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes a TTNA protospacer adjacent motif (PAM) distinct from the canonical TTTV PAM but with equivalent cleavage ability and specificity. These features significantly broadened the targeting scope of Cas12a family. Furthermore, we developed a sensitive, accurate, and rapid human papillomavirus (HPV) 16/18 gene detection platform based on LtCas12a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) and lateral flow assay (LFA). LtCas12a showed comparable sensitivity to quantitative polymerase chain reaction (qPCR) and no cross-reaction with 13 other high-risk HPV genotypes in detecting the HPV16/18 L1 gene. Taken together, LtCas12a can broaden the applications of the CRISPR-Cas12a family and serve as a promising next-generation tool for therapeutic application and molecular diagnosis.

SUBMITTER: Chen Y 

PROVIDER: S-EPMC10162949 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature