Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool

Clinical pathogen diagnostics detect targets by qPCR (but with low sensitivity) or blood culturing (but time-consuming). Here we leverage a dual-stem-loop DNA amplifier to enhance non-specific collateral enzymatic cleavage of an oligonucleotide linker between a fluophore and its quencher by CRISPR-CasΦ, achieving ultrasensitive target detection. Specifically, the target pathogens are lysed to release DNA, which binds its complementary gRNA in CRISPR-CasΦ to activate the collateral DNA-cleavage capability of CasΦ, enabling CasΦ to cleave the stem-loops in the amplifier. The cleavage product binds its complementary gRNA in another CRISPR-CasΦ to activate more CasΦ. The activated CasΦ collaterally cleaves the…

• NN
  National Natural Science Foundation of China

  Awards: 41866002, 42166001, 42306225, 42206212
• HU
  Hainan University

  Award: XTCX2022HYB09
• NN
  National Natural Science Foundation of China-Yunnan Joint Fund

  Award: 42376184