Team:ZJUT China B/Description
Description
Inspiration
With the development of molecular biology and synthetic biology, molecular diagnosis is moving towards the direction of increasing sensitivity, specificity, accuracy and detection speed. The continuous emergence of detecting methods like fluorescence quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and colloidal gold based immunoassay etc. facilitates the progress of the detection field.
At the end of 2019, a terrible virus called SARS-CoV-2 attacked the world. Due to the highly infectious feature of the SARS-CoV-2, cross infection has become a big threat to human society. From the history of virus-infected incidents, we really recognized that early detection and early prevention is of great significance to human health. Therefore, Point-of-Care Testing (POCT) may be the efficient solution to deal with the tricky issue. Based on that, we set up to make a highly efficient and portable detecting device which could target multiple viruses at the same time, thus to deal with more viruses and situations in the future.
CRISPR Cas13
CRISPR Cas13 protein is the effector of clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated genes (Cas) adaptive immune systems of microorganisms, which was discovered in 21 bacterial genomes. It's an RNA-guided RNase which could produces multiple cleavage sites in single-stranded areas of an target RNA with the preference of its corresponding crRNA. When the target exists in the sample, the crRNA will guide the Cas13 protein to find the target, and the collateral cleavage of Cas13 protein will be activated which means it could cleave bystander RNAs. With this feature, Cas13 protein could be used to detect and edit RNA.
The Cas13 protein encompasses four divergent family members (Cas13a–d). Different Cas13 protein homologue has different cleavage base preference.
According to this, we designed four kind fluorescence reporters to make the detecting result could be read out. (The base preference and the information of fluorescence reporter are shown in the Table 1)
Figure 1 The principle of fluorescence detecting
Multivirus Monitor
Our project covers the whole process from sample processing to the read out of detecting results.
As for sample processing, we use thermal lysis and Recombinase Polymerase Amplification (RPA) to release the viral nucleic acid and amplify the viral signal. And we designed detecting device as the application of our project. Besides, we designed user oriented interactive platform and database to make detecting results visible to users.
Ultimately, we integrated our project and named it as "Multivirus Monitor". It consists of three aspects: viralertor, virusee and viralibrary.
Figure 2. The work flow of detecting process
Viralertor
Viralertor is our detecting device which could detect multiple viruses in one pot. It encompasses three parts, the detector for fluorescent signals' reading; the microfluidic chip for samples processing; and the detection chip for converting nucleic acid signals into fluorescent signals. The detecting result could be viewed through electronic terminals. As the core of our project, the viralertor has highly efficient, portable and multivirus features, which is a potential weapon against viruses, old and new.
Figure 3. 3D demonstration of the detector
Viralibrary
The "viralibrary" is a crRNA database that we designed for basic scientific researchers to make it easy for obtaining crRNA from viruses. Since crRNA is composed of two parts ( DR sequence and Spacer, DR sequence is fixed and Spacer can be changed according to the retrieved virus ), users need to choose Cas13 protein and input with the virus name to obtain the complete sequence of crRNA.
Figure 4. Viralibrary
Virusee
The “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.
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. doi: 10.1038/s41596-019-0210-2. Epub 2019 Sep 23. Erratum in: Nat Protoc. 2020 Mar;15(3):1311. PMID: 31548639; PMCID: PMC6956564.
[2]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.
[3]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.
[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. doi: 10.1126/science.aam9321. Epub 2017 Apr 13. PMID: 28408723; PMCID: PMC5526198.
[5]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.
[6]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.
[7]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.
[8]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.
[9]Ferreira CES, Guerra JCC, Slhessarenko N, Scartezini M, Franca CN, Colombini MP, Berlitz F, Machado AMO, Campana GA, Faulhaber ACL, Galoro CA, Dias CM, Shcolnik W, Martino MDV, Cesar KR, Sumita NM, Mendes ME, Faulhaber MHW, Pinho JRR, Barbosa IV, Batista MC, Khawali C, Pariz VM, Andriolo A. Point-of-Care Testing: General Aspects. Clin Lab. 2018 Jan 1;64(1):1-9. doi: 10.7754/Clin.Lab.2017.170730. PMID: 29479878.
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