Beyond its extraordinary genome editing ability, the CRISPR-Cas system has opened a new era of biosensing applications due to its high base resolution and isothermal signal amplification. However, the reported CRISPR-Cas sensors are largely only used for the detection of nucleic acids with limited application for non-nucleic acid targets. To realize the full potential of the CRISPR-Cas sensors and broaden their applications for detection and quantitation of non-nucleic acid targets, we herein report CRISPR-Cas12a sensors that are regulated by functional DNA (fDNA) molecules such as aptamers and DNAzymes that are selective for small organic molecule and metal ion detections. The sensor is based on the Cas12a dependent reporter system consisting of Cas12a, CRISPR RNA (crRNA) and its single stranded DNA substrate labeled with a fluorophore and quencher at each end (ssDNA-FQ), and fDNA molecules that can lock a DNA activator for Cas12a-crRNA, preventing the ssDNA cleavage function of Cas12a in the absence of the fDNA targets. The presence of fDNA targets can trigger the unlocking of the DNA activator, which can then activate the cleavage of ssDNA-FQ by Cas12a, resulting in an increase of the fluorescent signal detectable by commercially available portable fluorimeters. Using this method, ATP and Na + have been detected quantitatively under ambient temperature (25 °C) using a simple and fast detection workflow (two steps and <15 min), making the fDNA-regulated CRISPR system suitable for field tests or point-of-care diagnostics. Since fDNAs can be obtained to recognize a wide range of targets, the methods demonstrated here can expand this powerful CRISPR-Cas sensor system *
The antiviral cGMP-AMP (cGAMP)-imulator of terferonenes (STING) pathway is well characterized in mammalian cells. However, whether this pathway also plays a role in insect antiviral immunity is unknown. In this study, we found that cGAMP is produced in silkworm () cells infected with nucleopolyhedrovirus (NPV). In searches for STING-related sequences, we identified , a potential cGAMP sensor in We observed that overexpression effectively inhibits NPV replication in silkworm larvae, whereas dsRNA-mediated knockdown resulted in higher viral load. Cleavage and nuclear translocation of BmRelish, a NF-κB-related transcription factor, was also observed when BmSTING was overexpressed and was enhanced by cGAMP stimulation or viral infection of larvae. Moreover, we identified a caspase-8-like protein (BmCasp8L) as a BmSTING-interacting molecule and as a suppressor of BmSTING-mediated BmRelish activation. Interestingly, cGAMP stimulation decreased BmCasp8L binding to BmSTING and increased BmRelish activity. Of note, an interaction between death-related ced-3/Nedd2-like caspase (BmDredd) and BmSTING promoted BmRelish cleavage for efficient antiviral signaling and protection of insect cells from viral infection. Our findings have uncovered BmSTING as a critical mediator of antiviral immunity in the model insect and have identified several BmSTING-interacting proteins that control antiviral defenses.
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