In the proposed study, we will develop a paper-based colorimetric CRISPR biosensor to detect microbial indicator bacteria (genericE. coli) in agricultural water. There are three major steps including: preparation of aggregated AuNPs, the development of CRISPR systems, and the combination of colorimetric CRISPR-based detection of genericE. coli. Firstly, gold nanoparticles (AuNPs) will be synthesized and modified with single-stranded DNA (ssDNA). Based on the specific design, ssDNA-modified AuNPs will be crosslinked by other ssDNA, resulting in the color change from red to purple. Secondly, a CRISPR-Cas12a system will be developed to target DNA fromE. colicells. After cleaving the target DNA, the CRISPR-Cas12a system will be activated to cleave ssDNA that is modified on the surface of AuNPs. Because of the ssDNA degradation, the interparticle distances among the AuNPs will increase, resulting in the color change from purple back to red. Based on the red intensity, the target DNA fromE. colican be determined. Finally, the colorimetric CRISPR-based detection system will be conducted on a paper-based substrate to provide a field-deployable detection ofE. coli.Objective 1: Preparation of ssDNA-modified AuNPsThe aggregation and dispersion behavior of AuNPs will change the interparticle distances, exhibiting unique optical properties that will be clearly identified with the naked eye.10-11Aggregation of AuNPs will shift the absorption peak to longer wavelength and change the solution color from red to purple. In this objective, we will prepare ssDNA-modified AuNPs that will be aggregated by introducing another complementary ssDNA. In our previous studies, we have prepared AuNPs with a size of 2 or 30 nm in diameter.A commercially synthesized ssDNA will easily bond to AuNPs through an Au-S bond.ThessDNA on the surface of AuNPs will protect the aggregation of AuNPs, remaining the solution color of red. After introducing a specific ssDNA that can hybridize with the ssDNA on the surface of AuNPs, the interparticle distances among the AuNPs will decrease, resulting in the aggregation of AuNPs and the solution color change from red to purple.Objective 2: Development of a CRISPR-Cas12a system to detect DNA in bacteriaThe CRISPR technology has the ability to precisely and efficiently recognize target DNA in millions of bacterial gDNA, allowing it to become a popular genome-editing tool. In our previous report, a multiplexed CRISPR-Cas12a system has been successfully used to editClostridium difficile.In addition, a CRISPR-Cas12a system has been used to detect pUC19 plasmids and pUC19 plasmid-positive bacteria (see the preliminary result). In this study, we will develop a programmable CRISPR-Cas12a system to detect DNA in genericE. coli. To optimize detection performances using the CRISPR-Cas12a system, we will explore at least 5 potential target bacteria gDNAs from the conservative regions inE. colicells. The one that produces the highest readout signal will be selected for future studies.Objective 3: Validation of the colorimetric CRISPR biosensor to detectgenericE. coliThanks to the colorimetric readout that is visible to the naked eye, AuNPs-based colorimetric assay without any sophisticated equipment is an attractive platform to detect microbial indicator bacteria. Combining with the programmable CRISPR-Cas12a system, we will develop a colorimetric CRISPR biosensor to detectE. coliin agricultural water. Briefly, the target DNA-activated CRISPR-Cas12a system can cleave the ssDNA among the aggregated AuNPs to increase the interparticle distances. The larger interparticle distances will shift the absorption peak back to the short wavelength, allowing the naked-eye detection ofE. coliin agricultural water. The detection sensitivity and specificity of genericE. coliwill be confirmed using a UV-vis spectrometer.Objective 4: Fabrication of a paper-based colorimetric CRISPR biosensor to detect genericE. coliRecently, paper matrices have been widely used as a substrate for bioassays due to their low-cost, disposability, and ease of use. Paper is a cellulose fiber web with a lot of microstructures that can provide a strong capillary force for the liquid flow. Also, the liquid flow can be controlled by printing hydrophobic materials into the hydrophilic paper matrix.The developed colorimetric CRISPR assays will be conducted onthe paper-based substrate. GenericE. coliin agricultural water will be determined by observing the color change and the results will be analyzed using the naked eye. To detectE. colicells at 10 or 100 CFU/mL in agricultural water, isothermal DNA amplification technologies will be used to amplify the target DNA, including loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA).