CRISPR/Cas12a-based dual amplified biosensing system for sensitive and rapid detection of polynucleotide kinase/phosphatase

• Published in: Biosensors & bioelectronics 2020-11, Vol.168, p.112556-112556, Article 112556
• Main Authors: Wang, Dong-Xia, Wang, Jing, Du, Yi-Chen, Ma, Jia-Yi, Wang, Si-Yuan, Tang, An-Na, Kong, De-Ming
• Format: Article
• Language: English
• Subjects: Biosensing; Biosensing Techniques; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas Systems - genetics; CRISPR/Cas12a; Nicking enzyme; Phosphoric Monoester Hydrolases; Polynucleotide 5'-Hydroxyl-Kinase - genetics; Polynucleotide kinase/phosphatase (PNKP); Strand displacement amplification
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2020.112556

We reported a CRISPR/Cas-based dual amplified sensing strategy for rapid, sensitive and selective detection of polynucleotide kinase/phosphatase (PNKP), a DNA damage repair-related biological enzyme. In this strategy, a PNKP-triggered nicking enzyme-mediated strand displacement amplification reaction was introduced to enrich the activator DNA strands for CRISPR/Cas. Such an isothermal DNA amplification step, together with subsequent activated CRISPR/Cas-catalyzed cleavage of fluorescent-labeled short-stranded DNA probes, enable synergetic signal amplification for sensitive PNKP detection. The proposed strategy showed a wide linear detection range (more than 3 orders of magnitude ranging from 1× 10−5 to 2.5 × 10−2 U/mL T4 PNKP) and a detection limit as low as 3.3 × 10−6 U/mL. It was successfully used for the PNKP activity detection in cell extracts with high fidelity and displayed great potential for enzyme inhibitor screening and inhibitory capability evaluation. This work broadens the applications of CRISPR/Cas12a-based sensors to biological enzymes and provides a way to improve the sensitivity by introducing an isothermal signal amplification step. Such an isothermal DNA amplification-CRISPR/Cas-combined biosensor design concept might expand CRISPR/Cas-based sensing systems and promote their applications in various fields such as disease diagnosis and drug screening. •A CRISPR/Cas12a-based sensing method is reported for polynucleotide kinase/phosphatase (PNKP) activity assay.•The method enables highly sensitive detection of T4 PNKP with a detection limit of 3.3 × 10−6 U/mL.•This work broadens the applications of CRISPR/Cas12a-based sensors to biological enzymes.•The proposed dual amplification strategy provides a way to improve the sensitivity of CRISPR/Cas-based sensors.•The method is demonstrated to work well for complex biological samples.