The extensive utilization of CRISPR-Cas systems in molecular diagnostics stems from their crRNA-guided transcleavage capabilities. However, AsCpf1-based detection systems frequently exhibit unexplained sensitive variations. This research reveals that AsCpf1 maintains a crRNA-independent function, similar to exonuclease I, when utilized in standard buffers containing Mg2+. From a structural perspective, this exonuclease activity is independent of the RuvC domain-mediated canonical trans-cleavage activity. It is predicted by structural modeling to be potentially localized within the WED-PI domain. In the context of diagnostics, the effective target concentration is diminished by AsCpf1-mediated degradation of the free 3 ' ends of target DNA, which impairs detection sensitivity. To mitigate this interference, we demonstrate that 3 ' end capping effectively restores detection performance. This approach was validated in a CRISPR-EXPAR-based microRNA biosensor, which exhibited approximately 10-fold improvement in sensitivity following 3 ' end capping. Overall, this investigation characterizes a previously unidentified exonuclease activity within the AsCpf1 system and establishes practical design criteria to improve the robustness and accuracy of CRISPR-based diagnostic tools.
[1]Jilin Univ, China Japan Union Hosp, Dept Oncol & Hematol, Changchun 130031, Jilin, Peoples R China.
[2]Chinese Acad Agr Sci, Changchun Vet Res Inst, State Key Lab Pathogen & Biosecur, Changchun 130122, Jilin, Peoples R China.
[3]Zhengzhou Univ, Affiliated Hosp 1, Dept Neurol, Zhengzhou 450052, Henan, Peoples R China.
[4]Nanjing Agr Univ, Coll Vet Med, Key Lab Anim Microbiol, Chinas Minist Agr, Nanjing 210095, Peoples R China.
[5]Dalian Med Univ, Affiliated Hosp 1, Cardiovasc Radiol Dept, Dalian 116021, Liaoning, Peoples R China.
(8.0+极速模式)