Team:ZJUT China B/Poster

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Poster: ZJUT China B

In the poster, you can have a comprehensive understanding of who we are, what we did, and how we did it. Enjoy exploring!
(Please click on the poster to view the content of each section )

Multivirus Monitor

Presented by ZJUT_China_B

Abstract:

The global pandemic of COVID-19 highlights the great importance of detecting viruses. We aim to construct a "Multivirus Monitor" as an application of CRISPR Cas13 in point-of-care diagnostics, which could detect multiple viruses in one pot. We tested the feasibility of the platform by testing the specificity between Cas13 orthologs and their sequence-specific reporters. We designed a multivirus detecting device which combines sample processing, amplification and detecting chambers. Based on fluorescence detecting, the detection results can be processed and transmitted to mobile terminals to make the results visible. We are designing an interactive platform called "virusee" which could provide optimal solutions and suggestions to users. Furthermore, we are constructing a database called "viralibrary", which could provide users with viral information and its corresponding crRNA. Conceived as a multivirus detecting and feedback platform for rapid point-of-care diagnostics, Multivirus Monitor can be a promising weapon in the unpredictable fight against viruses.

Team Roster:

PI: Xiaoling Tang; Feng Cheng
Advisor & Instructor: Asal Golshaie; Yuanshan Wang; Dongchang Sun; Jingyan Ge
Team members: Xiaojie Zhou; Cihui Lin; Yuzhi Wang; Yi Fan; Jiangxiaojie Zhang; Runlin Hou; Lu Wang; Xiaoqi Wang; Xinwei Wang; Xuran Zhao; Qian Rong; ZiYan Huang; Chaonan Fang; Xinzhi Zhou; Mengying Wang; Yuanying Wang; Chen He

Background

The data of confirmed cases and deaths of COVID-19
(Statistics by November 9, 2020)

At the end of 2019, SARS-CoV-2 hit the world, resulting in a global pandemic and millions of deaths. Because of the infectivity of COVID-19, cross infection has become a thorny problem. People suspected of infection cannot be treated because they cannot be diagnosed, and the lack of detection kits makes the diagnosis very difficult. COVID-19 has become a global pandemic with an increasing number of infections, resulting in 1.2 million deaths and the number is still increasing.

Inspiration & Goals

Inspiration

When our team was set up, we were at home, worrying about the situation of COVID-19 every day. We all wanted to do something to fight against the virus. Finally, we chose multivirus detection as our project, hoping our efforts could help human beings fight against the virus, not only now, but also in the future.
Point-of-Care Testing (POCT) may be the efficient solution to deal with the tricky issue because of its breaking of laboratory restrictions. After consulting the literature, we realised that rapid, inexpensive, sensitive and multiplexed nucleic acid detection may aid point-of-care pathogen detection.
Based on this, we designed a questionnaire to get an inner look at the public attitude towards portable multi-virus detectors.

Background research

Goals

1. Make a portable, accurate, inexpensive, easy to operate virus detection device, so that ordinary people can detect at home, solve the problem of cross infection.
2. Expand the number of viruses that can be detected by the detector at one time, and return the determined virus detection results to improve the efficiency of virus detection.
3. Establish an integrated detection system from sampling to result reading and terminal feedback.

Mechanism

Mechanism of Cas13 protein cleavage activity

Collateral cleavage

Cas13 protein is a single-component programmable RNA-guided RNA-targeting CRISPR effector, which belongs to class 2 typeVI CRISPR Cas system. It exhibits endoRNase activity mediating ssRNA cleavage with a single crRNA guide and cleaves bystander RNAs in addition to crRNA-targeted ssRNA (known as "collateral cleavage").

Cleavage base preference

Cas13 protein encompasses 4 divergent family members (Cas13a-d), different Cas13 protein homologue could produce multiple cleavage sites in single-stranded areas of an RNA target with different specific base preferences, which enables us detect multivirus in one pot.

Table. The base preference of Cas13 protein homologues

Fluorescence read out

We chose FRET fluorescent probes. It consists of a fluorophore on the one end and a quencher on the other, and the linking oligonucleotide varies according to the protein's base preference. The fluorescence is quenched when the probe is intact, when the linking oligonucleotide is cut off by Cas13 protein, the fluorescence will be emitted.

Sample processing

Viral plasmid

In order to simulate the natural state of the virus sequence wrapped in the protein shell, we constructed a plasmid with a short sequence of the target virus, and transferred it to E. coli to transcribe into RNA, and then obtained detectable target RNA from E. coli cell samples. In that way, the sample processing unit could be closer to the real state.

Pyrolysis

Pyrolysis can easily and quickly break E. coli cells and release target RNA. Then we use Trizol to extract RNA from the lysate, which can quickly break cells and inhibit the nuclease released by cells, and can maintain the integrity of RNA when disrupting and lysing cells.

Recombinase polymerase amplification (RPA)

Recombinase polymerase amplification (RPA) is known as an isothermal nucleic acid amplification technology. Its optimal temperature is between 37°C and 42°C. It can be carried out at room temperature without denaturation, which can truly realize portable and rapid nucleic acid detection. We combine reverse transcription (RT) with RPA to reversely transcribe the extracted target RNA into a DNA vector for amplification. In addition, since Cas13 targets RNA instead of DNA, the amplified DNA is transcribed into RNA by vitro transcription.

Cas13 protein

To obtain these proteins with high purity and activity, we designed biobricks to express the four Cas13 proteins homologues. We attached 6×His/Twinstrep tag to make Cas13 protein be purified by Ni-NTA purification. Besides, we added SUMO/MBP tag to increase the solubility of the proteins and prevent Cas13 proteins from forming inclusion bodies. These biobricks are under control of T7-promoter and Tphi-Terminator to maximize the protein's expression.

Expression and purification of Cas13

The workflow of Cas13 protein expression and purification

The 6xHis tag allows the expressed Cas13 protein to be purified by Ni-NTA purification, and the purification result can be checked by SDS-PAGE.
Regarding SUMO/MBP tag's potential effect on the activity of Cas13 proteins, the tag could be removed by SUMO protease or TEV protease, and the digested products need a second purification process to remove the digested fragments.

Detecting

Fluorephore

Considering the crosstalk between different fluorephors' excitation and emission wavelength and the quenching effect of corresponding quencher, Cy5, FAM, VIC, Texas-Red-X were finally chosen to be used in our detecting system, which allows the result to be detected because of their emitted fluorescence with different colors.

Linking oligonuleotide

The linking oligonucleotide is changed to AU, AC, GA, UC based on the base preference. Each fluorescent reporter is made to corresponds to Cas13 proteins as well as specific viral target sequence. We set up parameters of fluorescence plate reader according to the wavelength of our chosen filters to prove our concept.

Final fluorescent reporters

Human Practices to Wet Lab

Probe design with GenScript

With the help of GenScript, we were able to consider the probe structure, synthesis path, and price. Finally, after four versions of improvement, we got the suitable fluorescent probe.

Interview with professors

Viralertor

Introduction

We designed a device for the project called "Viralertor". Its detection unit is composed of LED light source, filter discs, sample plate and detection chip. It can detect different fluorescences by changing the filters.

After going through the Filter disc 1, the light can be filtered into the wavelength that can excite fluorescein to produce fluorescence. And the Filter disc 2 removes the light produced by Filter disc 1, only the light emitted by fluorescein can be detected by the sensor.

Demonstration

While there is only Cy5 in the sample, we can distinctly observe the flourescence of Cy5 (Lower right). And in other channels there is barely light which proves the feasibility of "Viralertor".

Sample processing

We also designed microfluidic chip and detection chip in order to simplify the complex operations. The microfluidic chip contains a sample processing unit ( include pyrolysis, cooling ) and a RPA unit ( recombinase polymerase amplification ). The detection chip can complete the transformation from nucleic acid signal to fluorescent signal.

Detection chip and microfluidic reactor

History

Our device has undergone several generations of improvement, and eventually, at BIOER's suggestion, we chose to use a pair of filters for detection. It's the filters sponsored by BIOER that makes the implementation of our device possible.

Our reactor has also undergone some improvements. At first we wanted to use paper chips to realize microfluidics, but the effect of controlling liquids in this way was not ideal. Later, we referred the device designed by Munich 2017, and designed such a microfluidic reactor. It uses a syringe as a power source. Theoretically, the liquid can be stationary at any position for reaction, such as RPA amplification, pyrolysis, etc.

Human Practices to Dry Lab

BIOER Company

The technical director of BIOER suggested that we add a pair of filters at the incoming light and the outgoing light to get a cleaner filter in order to reduce the influence of LED scattering, which was taken into account in the design of our fourth-generation device. In addition, BIOER sponsored us with four sets of expensive filters.

Demonstration

Cloning

The result of colony PCR confirmed C1,C2,C6,C8,C10 as positive colonies, which suggests that our construction is successful.

Cas13 protein homologues expression and purification

The results suggest that the protein was successfully expressed, and the obtained protein could be used in the detecting experiments, although it's not completely purified.

Fluorescence detection

Collateral cleavage activity and base preference of Cas13

Both LwaCas13a and PsmCas13b exhibit collateral cleavage activity. The base preference is redefined as "the activating associated cleavage base preference" based on our results and the base preference of Lwa and Psm is AU and GA respectively. After normalizing the data, although the influence of background fluorescence was minimized, a negative control is also needed to tell the detecting results.

Model

Equations and parameters

Complex: Cas-crRNA-target compound, activated Cas13 Lwa protein

Results

Through experiments, we fitted the parameters related to the equation. And by simulating the different k values (Figure 1), we found that the decrease of k value will reduce fluorescence peak value and extend peak time. The decrease in protein activity is directly reflected in the k value. Although lyophilization does facilitate the preservation of the protein, it reduces the detection efficiency, which is a big problem. Thus, we collaborated with QHFZ-China and tried to use TDPs (Tardigrade intrinsically Disordered Proteins) to make up for the activity loss due to lyophilization.

Figure 1. Scanning based on the fitted data

Implementation

Viralertor

Virusee

Viralibrary

Education

Classroom on cloud

Freshman Seminar

Summer social practice activity

For different courages' students, we participated in the summer social practice held by the school to publicize the relevant knowledge of synthetic biology and the preliminary ideas of our project

Wechat official account and Bilibili

Fun Game

Collaborations

QHFZ-China:

SJTU-Soft:

Other teams:

Communications

Innovation

There are already many nucleic acid based detection methods that have been put into use. Compared with these detection methods, we think our project has the following innovations.

1. Enable detection of multiple viruses in one pot. 
2. RPA makes the amplification process possible at room temperature.
3. The detection device, Viralertor, displays high efficiency, convenience and sensitivity.
4. Viralertor, Virusee, Viralibrary provide a integrated virus detection platform, making the detecting result feasible, enabling our project to meet the needs of a universal range of users.

Future

Due to the impact of COVID-19, not all our designs and plans have been completed on time. But we believe our project has potential for improvements. These are some of the future directions we suggest.

Project

1. Combining sample processing results with detection;
2. Improving the protocols of Cas13 protein purification;
3. Moving solubility tags of four expressed cas13 proteins;
4. Further Verifying the cleavage base preference;
5. Exploring the detection limits and specificity;
6. Optimizing the concentration of system components.

Application

1. Optimizing the lyophilyzation with TDPs and on the chip;
2. Exploring how to return more accurate detecting results from fluorescence signal;
3. Combining the sample processing microfluidic chip and the detection chip to make viralertor smaller;
4. Solving the problem of medical waste recycling;
5. Optimizing the feedback system of Virusee;
6. Setting up a negative control chip, test synchronously with the sample, and comparing the results between them;
7. Using glass fibrous media in detection chip to obtain better detection results.

Acknowledgements

References

[1]Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019 Oct;14(10):2986-3012.
[2]Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018 Apr 27;360(6387):439-444.
[3]Ackerman CM, Myhrvold C, Thakku SG, Freije CA, Metsky HC, Yang DK, Ye SH, Boehm CK, Kosoko-Thoroddsen TF, Kehe J, Nguyen TG, Carter A, Kulesa A, Barnes JR, Dugan VG, Hung DT, Blainey PC, Sabeti PC. Massively multiplexed nucleic acid detection with Cas13. Nature. 2020 Jun;582(7811):277-282.
[4]Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 Apr 28;356(6336):438-442.

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