Rapid and reliable nucleic acid detection methods are essential in clinical diagnostics and biotechnology.The clustered regularly interspaced short palindromic repeats(CRISPR)system is emerging as a next-generation nu...Rapid and reliable nucleic acid detection methods are essential in clinical diagnostics and biotechnology.The clustered regularly interspaced short palindromic repeats(CRISPR)system is emerging as a next-generation nucleic acid detection technology,offering versatility,convenience and rapid detection.However,CRISPR methods are significantly limited by the protospacer adjacent motif(PAM)sequence,and achieving a one-pot reaction for detecting single nucleotide variations(SNVs)within a short time still remains challenging.Here,we developed a comprehensive method for screening PAM sequences,which significantly expands the CRISPR detection scope.Additionally,we also proposed a one-pot CRISPR method,termed"SIMPLE",capable of identifying SNVs within 30 min.We applied the SIMPLE method to the clinical diagnostics of drug-resistant bacteria and the screening of cancer hotspot mutations.The SIMPLE method successfully detected drug-resistant bacteria mediated by canonical PAM TTN sequence with a sensitivity of 10 copies per reaction and achieved 100%consistency with next-generation sequencing results.Furthermore,the SIMPLE method proved effective in detecting hotspot mutations in cancer,even at a low mutation rate of 1%in the presence of high background interference mediated by non-canonical PAM ATN sequence.Therefore,the SIMPLE method not only expands the CRISPR detection scope but also offers a one-pot reaction with high specificity for SNVs identification,making it a promising tool for next-generation molecular diagnostics.展开更多
基金supported by the National Natural Science Foundation of China(32371521,82300220)Special Project for Experimental Animal Research(23141900300)+3 种基金Shanghai Rising Star Program(23QA1404300)Special Project for Medical Innovation Research(22Y11909200)Greater Bay Area Institute of Precision Medicine(Guangzhou)Human Phenome Data Center of Fudan University and Shanghai Municipal Science and Technology Major Project.(2023SHZDZX02).
文摘Rapid and reliable nucleic acid detection methods are essential in clinical diagnostics and biotechnology.The clustered regularly interspaced short palindromic repeats(CRISPR)system is emerging as a next-generation nucleic acid detection technology,offering versatility,convenience and rapid detection.However,CRISPR methods are significantly limited by the protospacer adjacent motif(PAM)sequence,and achieving a one-pot reaction for detecting single nucleotide variations(SNVs)within a short time still remains challenging.Here,we developed a comprehensive method for screening PAM sequences,which significantly expands the CRISPR detection scope.Additionally,we also proposed a one-pot CRISPR method,termed"SIMPLE",capable of identifying SNVs within 30 min.We applied the SIMPLE method to the clinical diagnostics of drug-resistant bacteria and the screening of cancer hotspot mutations.The SIMPLE method successfully detected drug-resistant bacteria mediated by canonical PAM TTN sequence with a sensitivity of 10 copies per reaction and achieved 100%consistency with next-generation sequencing results.Furthermore,the SIMPLE method proved effective in detecting hotspot mutations in cancer,even at a low mutation rate of 1%in the presence of high background interference mediated by non-canonical PAM ATN sequence.Therefore,the SIMPLE method not only expands the CRISPR detection scope but also offers a one-pot reaction with high specificity for SNVs identification,making it a promising tool for next-generation molecular diagnostics.
