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bioRxiv · 10.1101/2024.03.05.583466

Hairpin-locker mediated CRISPR/Cas tandem system for ultrasensitive detection of DNA without pre-amplification

Deng, F.·Sang, R.·Li, Y.·Yang, B.·Zhai, X.·Xue, R.·Zhang, C.·Deng, W.·Goldys, E. M.
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Abstract

Achieving ultra-sensitive detection of DNA is of paramount importance in the field of molecular analytics. Conventional amplification technologies such as polymerase chain reaction (PCR) currently play a leading role in ultrasensitive DNA detection. However, amplicon contamination common in these techniques may lead to false positives. To date, CRISPR-associated nucleases (type V & VI) with their programmable cleavage have been utilised for sensitive detection of unamplified nucleic acids in complex real samples. Nevertheless, without additional amplification strategies, the pM range sensitivity of such CRISPR/Cas sensors is not sufficient for clinical applications. Here, we established a hairpin-locker (H-locker) mediated Cas12-Cas13 tandem biosensing system (Cas12-13 tandem-sensor) for ultrasensitive detection of DNA targets. Without the need for any additional amplification reaction or device, this system is capable of detecting DNA at a notable 1 aM level (<1 copy/uL) sensitivity. In addition, the system was able to distinguish cancer mutations in colorectal cancer (CRC) mice. This is a significant advance for CRISPR/Cas biosensing technology offering simple, highly sensitive, and user-friendly diagnostics for next-generation nucleic acid detection. O_TBL View this table: org.highwire.dtl.DTLVardef@7fc32eorg.highwire.dtl.DTLVardef@1e716ceorg.highwire.dtl.DTLVardef@8a43e0org.highwire.dtl.DTLVardef@16e286dorg.highwire.dtl.DTLVardef@1202eb0_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONDNA and RNA oligos used in this study. C_TABLECAPTION C_TBL

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Deng, F., Sang, R., Li, Y., Yang, B., Zhai, X., Xue, R., Zhang, C., Deng, W., Goldys, E. M.. 2024-03-05. Hairpin-locker mediated CRISPR/Cas tandem system for ultrasensitive detection of DNA without pre-amplification. https://doi.org/10.1101/2024.03.05.583466

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