Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
DEVELOPMENT OF CRISPR-EQUIPPED ENGINEERED PHAGES (CREEPS) FOR THE DETECTION OF ESCHERICHIA COLI (INDICATOR BACTERIA) IN AGRICULTURAL WATER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1024643
Grant No.
2021-67017-33439
Cumulative Award Amt.
$450,462.00
Proposal No.
2020-03342
Multistate No.
(N/A)
Project Start Date
Nov 15, 2020
Project End Date
Nov 14, 2025
Grant Year
2021
Program Code
[A1332]- Food Safety and Defense
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Biological Systems Engineering
Non Technical Summary
The various ready-to-eat fresh produce is a potential vehicle for the transmission of foodborne pathogens. In the United States, over 1 million foodborne illnesses are caused by fresh produce contaminated with pathogens every year.Fresh produce microbial safety has been increasingly implicated as the second cause of foodborne illnesses as well as huge economic losses.Agricultural water has been evidenced as an important safety factor that leads to subsequent foodborne illness outbreaks.To improve fresh produce microbial safety, testing of agricultural waters has been required by the FDA Food Safety Modernization Act (FSAM). However, the most commonly used culture-based method to determine agricultural water microbial safety is time-consuming, taking at least 18 hours from samples to results. Other methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), are limited for the point-of-need (PON) applications due to the requirement of skilled operators and sophisticated equipment.The lack of a rapid and sensitive detection method that can be used by fresh produce workers to detect indicator bacteria in agricultural water is the current roadblock to fresh produce microbial safety.
Animal Health Component
30%
Research Effort Categories
Basic
20%
Applied
30%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71250101101100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
5010 - Food;

Field Of Science
1101 - Virology;
Goals / Objectives
In this project, we aim to develop a state-of-the-art CRISPR-equipped engineered phage (CREEP) for the rapid and sensitive detection ofEscherichia coli(E. coli) that serves as microbial contamination indicators in agricultural water. The cutting-edge detection assay will provide fresh produce workers with an easy-to-use tool to estimate the microbial safety of both irrigation and washing waters that come in direct contact with fresh produce. This is important for the long-term goal of preventing the spread of foodborne pathogens in the fresh produce supply chains and reducing foodborne outbreaks. The specific objectives include 1) design and optimize the CRISPR-Cas12a system to detect bacterial genomic DNA (gDNA), 2) fabricate CRISPR-equipped engineered phages (CREEP) to detect E. coli, and 3) validate CREEPs to estimate the microbial safety in agricultural water.
Project Methods
In the proposed study,phage T7 inserted with the CRISPR-Cas12a system will be developed as a proof-of-concept to detectE. colicells. Briefly, different DNA sequences in the conservative regions from the bacteria genome will be selected and characterized using the CRISPR-Cas12a system. The one that generates the highest readout signals will be selected as the target DNA. The DNA for complementary RNA (crRNA) and CRISPR Cas12a nuclease will be genetically engineered into the phage genome using molecular biology technologies. The CREEPs will then be employed to detect waterborne pathogens in agricultural water. The microbial safety of agricultural water (including irrigation water from growth farms and washing water from processing facilities) will be evaluated. (Aim 1)We will develop at least 10 CRISPR-Cas12a systems to detect the highest potential bacteria gDNA. Our group has already designed the CRISPR-Cas12a systems and will start to experimentally prepare these CRISPR-Cas12a systems for bacteria gDNA detection as below. (Aim 2)E. coli will be used as indicator bacteria because they are the most common bacteria on the earth as well as highly associated with agricultural water.We will engineerE. coli-specific phage T7 that can infect a broad range ofE. colicells.The DNA of Cas12a protein nuclease (requested fromaddgene.org) and the crRNA-complementary DNA that are optimized in Aim 1 will be inserted into the phage T7 genome. To enable the purification and characterization of the phage-introduced Cas12a-crRNA complexes, a polyhistidine tag will be introduced at the N-terminus of the Cas12a nuclease. (Aim3), The methods developed in this aimwill be similar to those described in Aims 1-2. The detection results can allow us to estimate the concentration ofE. colicells in agricultural water samples. In the future experiment,we will first detect the irrigation and washing waters spiked withE. colicells. The detection results of irrigation and washing waters in a blind study using T7CRISPRphages will be compared with the conventional culture- and molecular-based detection methods.

Progress 11/15/23 to 11/14/24

Outputs
Target Audience:It is important to share our findings and apply this technique in the fresh produce supply chain. During this period, we have reached other food scientists and fresh produce farmers through interactions at local,regional, and national conferences. For example, we presented our studies at a university-wide conference (center for emerging zoonotic, and arthropod-borne pathogens, CeZAP). Wehave contacted Fresh Produce Food Safety (FPFS) which aims to provide science-based resources for agricultural growers, farmers, and food handlers. In addition, we have have presented a talk and a poster at the national ACS Spring 2024 meeting. Wehave mentored twoundergraduate students and twograduate student who all havemajored in Biological Systems Engineering at Virginia Tech. One of these students has recieved a USDA predoctoral fellowship. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students (Yawen He and Tom kasputis, fourth-year Ph.D. students in the Department of Biological Systems Engineering at Virginia Tech) gave oral/poster presentations at national conferences, including International Association of Food Protection (IAFP) annual meeting, American Chemical Society (ACS), and American Society of Agricultural and Biological Engineers (ASABE) annual international meeting. The student was provided feedback by the communities of food safety and bioanalytical chemistry. Additionally, two undergrads were supported on this project to assist with molecular cloning. How have the results been disseminated to communities of interest?1)Presentations (Juhong Chen) and poster (Tom Kasputis) at ACS Spring 2024 in New Orleans 2)Oral presentations at the American Society for Agricultural and Biological Engineers and Center for Emerging, Zoonotic, and Arthropod-borne Pathogens Symposium (Tom Kasputis) 3)Oral presentation at American Society of Agricultural and Biological Engineers (ASABE) annual international meeting and poster presentation at the International Association for Food Protection (IAFP) annual meeting (Yawen He) What do you plan to do during the next reporting period to accomplish the goals?1) Publish 1-2 peer-reviewed papers. 2) Present findings at meetings (including the International Association of Food Protection, Gordon Research Conference, and American Chemical Society) to share the results with food scientists and food engineers who are working on food safety. 3) Work with undergraduate students, graduate students, and the fresh produce food safety (FPFS) program at Virginia Tech to develop a better understanding of the impacts of this study.

Impacts
What was accomplished under these goals? The various ready-to-eat fresh produce is a potential vehicle for the transmission of foodborne pathogens. Agricultural water (including irrigation water and washing water) has been evidenced as the main source to introduce foodborne pathogens to fresh produce. An ideal detection method would provide non-trained operators (including fresh produce growers on farms and food handlers in food processing facilities) with a rapid, sensitive, and low-cost tool to estimate the microbial contaminants (generic E. coli) in agricultural water. These abilities to detect microbial indicator bacteria will help us take immediate actions to prevent the spreading of foodborne pathogens in the fresh produce supply chain, including washing water and irrigation water. In this period, we have engineered phage T4 with a reporter gene (LacZ). In addition, we have applied the engineered phages to detect indicator bacteria in a buffer. The detection sensitivity and specificity have been determined.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Duan, M., Zhao, Y., Liu, Y., He, Y., Dai, R., Chen, J., et al. (2024). A low-background and wash-free signal amplification F-CRISPR biosensor for sensitive quantitative and visible qualitative detection of Salmonella Typhimurium. Science of The Total Environment 912, 168905. doi: 10.1016/j.scitotenv.2023.168905
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Tom Kasputis, Qiaoqiao Ci, Juhong Chen. On-site fluorescent detection of microbial contamination using a graphene oxide CRISPR-Cas12a (GO-CRISPR) sensor, Poster Board #112, ACS Spring 2024, March 17, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Juhong Chen. Development of phage-CRISPR nexus for microbial food safety. ACS Spring 2024. March 17, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yawen He, Xuemei Zhang, Juhong Chen. Simultaneous Dual-Gene Detection of Escherichia coli O157:H7 Based on CRISPR/Cas13-Mediated Biosensor. American Society of Agricultural and Biological Engineers (ASABE) annual international meeting, July 28, 2024, Anaheim, CA. (Oral Presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yawen He, Xuemei Zhang, Juhong Chen. Simultaneous Dual-Gene Detection of Escherichia coli O157:H7 Based on CRISPR/Cas13-Mediated Biosensor. International Association for Food Protection (IAFP) annual meeting, July 14, 2024, Long Beach, CA. (Poster Presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yawen He, Juhong Chen. CRISPR/Cas9 mediated genome editing of T4 bacteriophage for high-throughput antimicrobial susceptibility testing. UC Systemwide Bioengineering Symposium, June 24, 2024, San Deigo, CA. (Poster Presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: T. Kasputis, Y. He, J. Chen. On-site fluorescent detection of sepsis-inducing bacteria using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System, Oral presentation, Center for Emerging, Zoonotic, and Arthropod-borne Pathogens (CeZAP) Infectious Diseases Symposium. Oct. 11, 2024. Blacksburg, VA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: T. Kasputis, Y. He, J. Chen. On-site fluorescent detection of microbial contamination using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System. Oral presentation. American Society for Agricultural and Biological Engineers. Jul. 28-31, 2024. Anaheim, CA.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: T. Kasputis, Y. He, Q. Ci, J. Chen. On-site Fluorescent Detection of Salmonella using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System, ACS Analytical Chemistry, 2024, 96 (6), 26762683. doi: 10.1021/acs.analchem.3c05459


Progress 11/15/22 to 11/14/23

Outputs
Target Audience: It is important to share our findings and apply this technique in the fresh produce supply chain. During this period, we have reached other food scientists and fresh produce farmers through interactions at local conferences. For example, we presented our studies at a university-wide conference (center for emerging zoonotic, and arthropod-borne pathogens, CeZAP). I have contacted Fresh Produce Food Safety (FPFS) which aims to provide science-based resources for agricultural growers, farmers, and food handlers. In addition, I have mentored one undergraduate student and one graduate student who both majored in Biological Systems Engineering at Virginia Tech. I believe the students will rise up to the challenges of food safety and food security. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 1) One undergraduate student (Nopdanai Ramascoot,a senior year in the Department of Biological Systems Engineering at Virginia Tech) started to learn the CRISPR-based biosensor to detect foodborne pathogens. 2) One graduate student (Yawen He, a third-year Ph.D. student in the Department of Biological Systems Engineering at Virginia Tech) gave one poster presentation at the International Association of Food Protection (IAFP) annual meeting. The student was provided feedback by the communities of food safety and bioanalytical chemistry. How have the results been disseminated to communities of interest? 1) An oral presentation (Juhong Chen) orally presented research findings for this project to an audience of microbiologists at the annual American Chemistry Conference (ACS). 1) Oneposter presentation (Yawen he) orally presented research findings for this project to an audience of food microbiologistsatthe International Association of Food Protection (IAFP) annual meeting. What do you plan to do during the next reporting period to accomplish the goals?1) Publish 1-2 peer-reviewed papers. 2) Present findings at meetings (including the International Association of Food Protection, Gordon Research Conference, and American Chemical Society) to share the results with food scientists and food engineers who are working on food safety. 3) Work with undergraduate students, graduate students, and the fresh produce food safety (FPFS) program at Virginia Tech to develop a better understanding of the impacts of this study.

Impacts
What was accomplished under these goals? The various ready-to-eat fresh produce is a potential vehicle for the transmission of foodborne pathogens. Agricultural water (including irrigation water and washing water) has been evidenced as the main source to introduce foodborne pathogens to fresh produce. An ideal detection method would provide non-trained operators (including fresh produce growers on farms and food handlers in food processing facilities) with a rapid, sensitive, and low-cost tool to estimate the microbial contaminants (generic E. coli) in agricultural water. These abilities to detect microbial indicator bacteria will help us take immediate actions to prevent the spreading of foodborne pathogens in the fresh produce supply chain, including washing water and irrigation water. In this period, we have created aCRISPR-Cas9-mediated system to genetically engineer phages. Two plasmids (donor plasmid and pCas plasmid) can be co-transformedinto bacteria cells, in which the donor gene in the donor plasmid can be easily modified to engineer the phage genome with any DNA-of-interest. In order to improve the efficiency of the CRISPR-based detection of indicator bacteria, we have splitthe Cas12 into two parts. The split N-Cas12a and C-Cas12a have been inserted into the phage T4 genome, respectively.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Y. He, Q. Hu, S. San, T. Kasputis, M. Splinter, K. Yin, J. Chen. CRISPR-based biosensors for human health: a novel strategy to detect emerging infectious disease. TrAC Trends in Analytical Chemistry. 2023, Vol. 168 Pages 117342. DOI: https://doi.org/10.1016/j.trac.2023.117342.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Y. He, J. Chen. CRISPR/Cas9 mediated genome editing of T4 bacteriophage for high-throughput antimicrobial susceptibility testing. International Association of Food Protection Annual Meeting, 2023, July 16-19. Toronto Canada.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: T. kasputis, J. Chen. On-site fluorescent detection of Salmonella using a graphene-oxide CRISPR-Cas12a (GO-CRISPR) system. International Association of Food Protection Annual Meeting, 2023, July 16-19. Toronto Canada.


Progress 11/15/21 to 11/14/22

Outputs
Target Audience:It is important to share our findings and apply this technique in the fresh produce supply chain. During this period, we have reached other food scientists and fresh produce farmers through interactions at local conferences. For example, we presented our studies at a university-wide conference (Microbial Systems). I have contacted fresh produce food safety (FPFS) which aims to provide science-based resources for agricultural growers, farmers, and food handlers. In addition, I have mentored one undergraduate student and one graduate student who both majored in Biological Systems Engineering at Virginia Tech. I believe the students will rise up to the challenges of food safety and food security. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 1) One undergraduate student (Samantha San, a senior year in the Department of Biological Systems Engineering at Virginia Tech) practiced oral communication of technical results to an undergraduate research community at Virginia Tech. The student was provided feedback by the Fralin Life Science competition committee. 2) One graduate student (Yawen He, a second-year Ph.D. student in the Department of Biological Systems Engineering at Virginia Tech) gave two poster presentations at the Gordon ResearchConference (topic:Nanoscale Science and Engineering for Agriculture and Food Systems, andBioanalytical Sensors). The student was provided feedback by the communities of food safety and bioanalytical chemistry. How have the results been disseminated to communities of interest? 1) An undergraduate researcher (Samantha San) orally presented research findings for this project to an audience of undergraduate researchers at the summer seminar series for the Fralin Summer Undergraduate Research. 2) One graduate student(Yawen, a second-year Ph.D. student in the Department of Biological Systems Engineering at Virginia TechP gave two poster presentation to audiences in the field of nanotechnology for food safety and bioanalytical sensors for food safety. 3) An oral presentation (Juhong Chen) orally presented research findings for this project to an audience of food safety and food protection at IAFP. What do you plan to do during the next reporting period to accomplish the goals?1) Publish 1-2 peer-reviewed papers. 2) Present findings at meets (including the International Association of Food Protection, Gordon Research Conference, and American Chemical Society) to share the results with food scientists and food engineers who are working on food safety. 3) Work with undergraduate students, graduate students, and the fresh produce food safety (FPFS) program at Virginia Tech to develop a better understanding of the impacts of this study.

Impacts
What was accomplished under these goals? The various ready-to-eat fresh produce is a potential vehicle for the transmission of foodborne pathogens. Agricultural water (including irrigation water and washing water) has been evidenced as the main source to introduce foodborne pathogens to fresh produce. An ideal detection method would provide non-trained operators (including fresh produce growers on farms and food handlers in food processing facilities) with a rapid, sensitive, and low-cost tool to estimate the microbial contaminants (generic E. coli) in agricultural water. These abilities to detect microbial indicator bacteria will help us take immediate actions to prevent the spreading of foodborne pathogens in the fresh produce supply chain, including washing water and irrigation water. In this period, we have engineered phage T4 to express beta-galactosidase (beta-gal) to detect bacteria in food samples. The gene of beta-gal was inserted into phage T4 using the CRISPR-Cas9 system. The inserted gene was exchanged with a gene after capsid proteins by homologous recombination. The resulting phages were used to detect E. coli in abuffer solution. Currently, we are investigating the detection limit and specificity.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: ACS Food Sci. Technol. 2023, 3, 17-22
  • Type: Other Status: Accepted Year Published: 2022 Citation: Recent Advances in Phage-Based Systems


Progress 11/15/20 to 11/14/21

Outputs
Target Audience: It is important to share our findings and apply this technique in the fresh produce supply chain. During this period, we have reached other food scientists and fresh produce farmers through interactions at local conferences. For example, we presented our studies at a university-wide conference (Microbial Systems). I have contacted fresh produce food safety (FPFS) who aims to provide science-based resources for agricultural growers, farmers, and food handlers. In addition, I have mentored one undergraduate student and one graduate student who are both majored in Biological Systems Engineering at Virginia Tech. I believe the students will rise up to the challenges of food safety and food security in the future. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 1) One undergraduate student (Samantha San, junior year in the Department of Biological Systems Engineering at Virginia Tech) practiced oral communication of technical results to an undergraduate research community at Virginia Tech. The student was provided feedback by the Fralin Life Science competition committee. 2) One graduate student (Yawen He, first-year Ph.D. student in the Department of Biological Systems Engineering at Virginia Tech) practiced oral communication of technical results to a graduate seminar at Virginia Tech. The student was provided feedback by the graduate students. How have the results been disseminated to communities of interest? 1) An undergraduate researcher (Samantha San) orally presented research findings for this project to an audience of undergraduate researchers at the summer seminar series for the Fralin Summer Undergraduate Research. 2) An oral presentation (Juhong Chen) orally presented research findings for this project to an audience of microbiologists at the Microbial Systems annual conference at Virginia Tech. What do you plan to do during the next reporting period to accomplish the goals? 1) Publish 1-2 peer-reviewed papers. 2) Present findings at meets (including the International Association of Food Protection, Gorden Research Conference, and American Chemical Society) to share the results with food scientists and food engineers who are working on food safety. 3) Collect data related to objectives 2&3. 4) Work with undergraduate students, graduate students, and the fresh produce food safety (FPFS) program at Virginia Tech to develop a bettter understanding of the impacts of this study.

Impacts
What was accomplished under these goals? The various ready-to-eat fresh produce is a potential vehicle for the transmission of foodborne pathogens. Agricultural water (including irrigation water and washing water) has been evidenced as the main source to introduce foodborne pathogens to fresh produce. An ideal detection method would provide non-trained operators (including fresh produce growers on farms and food handlers in food processing facilities) with a rapid, sensitive, and low-cost tool to estimate the microbial contaminants (genericE. coli) in agricultural water. These abilities to detect microbial indicator bacteria will help us take immediate actions to prevent the spreading of foodborne pathogens in the fresh produce supply chain, including washing water and irrigation water. In objective 1, we have expressed and purified Cas12a nuclease. In addition, we havedesigned five CRISPR-Cas12a systems to detect bacterial genomic DNA and determined the one with the highest signal readout. The detection limit of extracted bacteria genomic DNA in the buffer solution has been determined using the optimized CRISPR-Cas12a system. In objective 2, we have produced a control T7 phage that cannot express the CRISPR-Cas12a system during phage infection. We plan to insert the gene of the CRISPR-Cas12a system into the phage.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: J. Chen. "Rapid Detection of Antibiotic-resistant Genes in the Critical Points of Food Supply Chain Using CRISPR-based Biosensors", Virginia Tech Microbial Systems. 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: S. San, and J. Chen. "Development of a CRISPR-assisted Lateral Flow Assay to Detect Foodborne Pathogens", Virginia Tech Fralin Life Science. 2020.