CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

TLDR

CRISPR‑Cas12a (Cpf1) is an RNA‑guided endonuclease that cleaves DNA and is employed for genome editing by generating targeted double‑strand breaks. The authors combine Cas12a ssDNase activation with isothermal amplification to develop DETECTR, a method achieving attomolar sensitivity for DNA detection. Cas12a shows indiscriminate single‑stranded DNA cleavage when bound to its target, a feature shared by other type V CRISPR‑Cas12 enzymes, and the DETECTR assay can rapidly and specifically detect human papillomavirus in patient samples.

Abstract

CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.