At the end of 2019,SARS-CoV-2 hit the world, arousing a global pandemic, causing millions of people to die. When our team was set up, we were at home, worrying about the situation of the COVID-19 every day.We all wanted to do something to fight against the virus. Finally, we choose multivirus detecting as our project, hoping our efforts could help human beings fight against the virus, not only now, but also in the future.
Our "Multivirus Monitor" is based on CRISPR Cas13 technology.To achieve our multivirus detecting goal, we chose four Cas13 proteins homologue as our detecting effectors, which were found in different organisms.The difference between them is their cleavage base preference, which enables us to make one to one detecting (One virus corresponds to one protein) in one pot even with the presence of multiple viruses.
We divided our project into 6 parts: sample processing, CRISPR Cas13 protein homologue expression and purification, fluorescence detecting, lyophilization and improvement, hardware and user oriented interactive platform and database. These parts covered all aspects that we could consider to make our project as perfect as possible. They will be separately completed at first, and finally they will be integrated into our "Muitivirus Monitor" to make the project more efficient to progress.
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, which brings the sample processing unit closer to the real state.
Thermal lysis can easily and quickly break E. coli cells and release target RNA.Then we use Trizol to extract RNA from the lysate. Trizol contains phenol, guanidine isothiocyanate and other substances, which can quickly break cells and inhibit the nuclease released by cells, and can maintain the integrity of RNA when disrupting and lysing cells. Therefore, the extraction of RNA and Purification is very useful.
Recombinase polymerase amplification (RPA) is known as a nucleic acid detection technology that can replace PCR. 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, which encompasses four divergent family members (Cas13a–d), is a RNA-guided RNase that produces multiple cleavage sites in single-stranded areas of an RNA target with specific base preferences. Cas13 also exhibits target-dependent promiscuous RNase activity, leading to transcleavage of bystander RNA molecules, an effect termed‘collateral cleavage’.
In order to achieve the purpose of multi-virus detection, we selected four Cas13 proteins, including Lwa, Lba of the Cas13a family and Psm, Cca of the Cas13b family because of their cleavage base preference.
We constructed biobricks and parts and attached it to plasmids to express Cas13 protein homologue.
The Cas13 protein expression plasmid contains the SUMO or MBP tag, which can prevent the large Cas13 protein from forming inclusion bodies and improve the solubility of the protein.
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 effect on the activity of Cas13 proteins, the tag could be removed by SUMO protease or TEV protease.
Based on the base preference of Cas13 protein homologue, we choose fluorescence as our read out format. 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 due to their emitted fluorescence with different colors. Then,by changing the linking oligo nucleotide to AU,AC,GA,UC,we made each fluorescence reporter corresponds to Cas13 proteins as well as specific viral target sequence. Other, in order to combine with the detecting device, we setted up parameters of fluorescence plate reader according to the wavelength of our chosen filters to prove our concept(table 1). That way, the result could be more convincing and more realistic.
The storage of Cas13 protein is the key to our application in the real world. To extend the shelf-life of our product, we designed to store and use Cas13 protein in a lyophilyzation form. However, the preservation of lyophilyzation protein will damage the activity of Cas13 protein, so we use Tardigrade intrinsically disordered proteins (TDPs) as an additive to lyophilyzation, which can reduce the effect of lyophilyzation on the activity of Cas13 protein.
We designed "viralertor" which could detect multiple viruses in a portable,cheap and visible way.
Because our project is very complicated, the sample needs to go through a lot of processing to get the final fluorescence signal. The fluorescence signal requires special equipment to be accurately read, otherwise the correct result will not be obtained due to the strong interference of the background light. Therefore, we divided the device into three parts: the detector, which is used to read fluorescent signals; the microfluidic chip, which is used to process samples; and the detection chip, which converts nucleic acid signals into fluorescent signals.The detecting result could be viewed through electronic terminals.
Regarding the implementation of our device,we designed "virusee" and "viralbrary" as our user oriented interactive media.
"viralibrary" is a crRNA database that we designed for basic scientific research personnel to make it easy for obtaining crRNA from viruses. Since crRNA is composed of two parts（DR sequence and Spacer）where DR sequence is fixed and Spacer changes according to the retrieved virus, the user needs to choose Cas13 protein and input with the virus name to obtain the complete sequence of crRNA.
“virusee” is a WeChat applet that we designed for the general public. We will strictly protect users' privacy and follow the principle of users' autonomy and willingness.We expect that “virusee” can read data from “viralertor” and have information query function at the same time, but we are not yet able to achieve synchronization with the alert data.Users can learn basic information about a virus by directly retrieving its name or corresponding symptoms. The basic information include susceptible population, protection, treatment, vaccine status and so on.
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. doi: 10.1038/s41596-019-0210-2. Epub 2019 Sep 23. Erratum in: Nat Protoc. 2020 Mar;15(3):1311. PMID: 31548639; PMCID: PMC6956564.
Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016 Aug 5;353(6299):aaf5573. doi: 10.1126/science.aaf5573. Epub 2016 Jun 2. PMID: 27256883; PMCID: PMC5127784.
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. doi: 10.1126/science.aaq0179. Epub 2018 Feb 15. PMID: 29449508; PMCID: PMC5961727.
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. doi: 10.1126/science.aam9321. Epub 2017 Apr 13. PMID: 28408723; PMCID: PMC5526198.
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. doi: 10.1038/s41586-020-2279-8. Epub 2020 Apr 29. PMID: 32349121; PMCID: PMC7332423.
Boothby TC, Tapia H, Brozena AH, Piszkiewicz S, Smith AE, Giovannini I, Rebecchi L, Pielak GJ, Koshland D, Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. Mol Cell. 2017 Mar 16;65(6):975-984.e5. doi: 10.1016/j.molcel.2017.02.018. PMID: 28306513; PMCID: PMC5987194.
Lobato IM, O'Sullivan CK. Recombinase polymerase amplification: Basics, applications and recent advances. Trends Analyt Chem. 2018 Jan;98:19-35. doi: 10.1016/j.trac.2017.10.015. Epub 2017 Oct 26. PMID: 32287544; PMCID: PMC7112910.
Zhu H, Richmond E, Liang C. CRISPR-RT: a web application for designing CRISPR-C2c2 crRNA with improved target specificity. Bioinformatics. 2018 Jan 1;34(1):117-119. doi: 10.1093/bioinformatics/btx580. PMID: 28968770.
Li Shijie,Yang Yankun,Liu Meng,Bai Zhonghu,Jin Jian.High-efficiency expression and purification of SUMO protease Ulp1 and preparation of scFv by His-SUMO tag[J].Chinese Journal of Bioengineering,2018,38(03):51-61.