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Coursework on grant proposal on the topic of CRISPR Cas9 and gene knockout....

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TAIBMS Research Development Fund Autumn 2021 Application Form Please complete the form using Arial 11 point and do not adjust the formatting of the application form. Use figures and tables to support your application. Completed applications should be uploaded via Turnitin before 9pm on 28th Oct 2021. ● FAQ: https://tinyurl.com/TAIBMSGrantFAQ2021 ● Library Endnote Guide: https://libguides.mmu.ac.uk/endnote Grant proposals must be written in your own words. Turnitin will be used to generate similarity reports, including similarity with other student’s proposals. Please indicate which topic you are applying for: ● You must submit an Dr Hargreaves - CRISPR-Cas to Solve the problem of application based on the Anti-microbial Resistance topic you were assigned. Applicant Details – all applicants should complete these questions Name

Polina Lobacheva

Description of Activities – all applicants should complete these questions ● This funding CANNOT be used to fund clinical trials. ● The proposal MUST include molecular biology techniques/assays. ● The proposed work should be achievable within 12 months. Title (max 25 words)

Knockout of antimicrobial resistance genes, rpsL and rrs, in multidrug-resistant tuberculosis using CRISPR Cas9.

Start Date (DD/MM/YYYY)

ASAP

Duration (Months)

12 months

Hypothesis (1 sentence)

My hypothesis is that the knockout of rpsL and rrs genes will prevent their translation from being inhibited and allow for the Streptomycin drug to be used successfully to treat MDR-TB. Objectives (Max 150 words) Please provide an overview of the aims & objectives for your project. Mycobacterium tuberculosis (Mtb), a pathogenic bacterium that results in tuberculosis (TB), latently infects around 2 billion people globally (Choudhary, 2015). It is estimated that three in 1000 people carry the latent Multi-Drug Resistance TB (MDR-TB) infection (Knight, 2019). Streptomycin, one of the first and most effective treatments for TB, is one of the 5 first-line drugs used to treat MDR-TB. However, Mtb became resistant to the antimicrobial agent due to a genetic mutation (Palomino, 2014) that inhibits the translation of rpsL and rrs genes (Smith, 2012). In 2017, MDR-TB caused an estimated 14% of TB deaths worldwide (Knight, 2019). This project is essential for the development of quicker and more effective treatments for those with MDR-TB. Objectives: 1. Design sgRNAs specific to rpsL and rrs genes 2. Knockout rpsL and rrs genes from Mtb using CRISPR Cas9 3. Validate successful gene knockout through arrays and qPCR Proposed Activities (Max 750) Please provide an overview of the experiments you propose to do. An estimated one third of the world is infected with Mtb bacteria that causes Tuberculosis (TB). The majority of those infected with Mtb are carrying the disease in dormant state and only 5-10% will develop TB in their lifetime (Abel, 2019). Streptomycin had a high success rate before MDR-TB developed microbial resistance to the drug. With resistance to newer drugs, previous antibiotics and treatments are being reanalysed (Cohen et al., 2020). CRISPR Cas9 is a gene editing tool that can knockout genes that are responsible for causing the antimicrobial resistance in MDR-TB. Knocking out the rpsL and rrs genes, responsible for Streptomycin resistance, will develop a more efficient treatment for MDR-TB. CRISPR Cas9 can precisely target specific genes in the sequence and successfully edit the sequence (Hille, 2016). Obtaining Mtb cells: In order to perform this research, it would be necessary to obtain Mtb bacteria cells from American Type Culture Collection (ATCC, 2021). CRISPR Cas9: Designing the single guide RNA (sgRNA) and finding the PAM sequence The CRISPR Gene Knockout Kit V2 will be used for this project and will be ordered from Synthego (Synthego, 2021). Using the NCBI “Gene” database, we will be able to obtain the DNA sequence for rpsL and rrs genes (NCBI, 2021). Consequently, the derived DNA sequence will be sent to Synthego to generate sgRNA for this experiment (CRISPRevolution sgRNA EZ Kit, 2021). The sgRNA, made up of a tracrRNA and crRNA, guides Cas9 to match the target specific gene site on the DNA to begin cleaving the double stranded break (DSB) in the genomic site (Collias, 2021).

Delivering the sgRNA and CAS9: Electroporation to insert Cas9 and sgRNA into the target cell sgRNA is cloned by inserting a specific plasmid into gRNA with the gene of interest, then the gRNA replicates itself into several clones. The expressed gRNA will be isolated from the plasmid and synthesised through in vitro transcription (Liu et al., 2020). The electroporation method is an effective way to deliver Cas9 reagents to the target cell, as it leads to a more immediate response and increases the efficacy of genetic changes (Lino, 2018). The electroporation procedure is further demonstrated in Figure 1. This method is efficient as it delivers to the whole cell population and is a well-known technique.

Knockout rpsL and rrs genes: The DNA break created by Cas9 is repaired by the cell’s non-homologous end joining NHEJ pathway, which introduces site specific deletions and insertions. (Tuladhar et al., 2019). The process is further demonstrated in Figure 2.

Validate the experiment: (1) Polyclonal screening: Using the Mismatch Cleavage Assay (Figure 3) and a surveyor nuclease, we will be able to determine the number of cells that have been genetically modified. The edited region is amplified by PCR, then DNA strands are denatured and reannealed (Qiu et al., 2004). The strands separate and randomly re-hybridise, creating heteroduplexes if editing occurred. In this case, the Surveyor nuclease will create a DSB at the site of a mismatch between two different strands of annealed DNA (Qiu et al., 2004). The cleaved DNA will run on an agarose gel suggesting the portion of the pool that has been edited through the presence of cleavage bands. The Mismatch Cleavage Assay is a quick and inexpensive method to test whether editing has occurred (Qiu et al., 2004).

(2) Isolate monoclonals:

Using a serial dilution method to isolate single cells from the pool of edited genes to create monoclonal cell lines (Alberts et al., 2002). (3) Monoclonal Screening Next Generation Sequencing (NGS) can identify a biallelic or homozygous mutation that results in a frameshift. A gene is fully knocked out if all the sequences show frameshift mutations at the region (Yang et al., 2017). The advantage of using NGS over Sanger sequencing is its ability to perform high-throughput screening of many monoclones simultaneously and can identify any off target editing that may have occurred during the CRISPR procedure (Yang et al., 2017). Validate the status of edited genes (Applied Biological Materials, 2021): Using two agar plates, we can check gene removal from Mtb. The surface of both plates would have a thin layer of Streptomycin. Plate 1 would be dotted using the wild type of Mtb and act as the control. Plate 2 would be covered in the same pattern using the newly edited Mtb If the cells on Plate 2 disappear then gene editing has been successful as the antibiotic killed the edited Mtb cells. Impact - potential for impact in your research field – explain how this work will make a contribution to your research field. What is the potential for future clinical impact? (100 words). A quarter of all deaths caused by antimicrobial resistant infections are a consequence of MDR-TB strains of Mtb (O’Neill, 2016). Patients suffering from MDR-TB are often faced with failed diagnosis, low success of treatment and an inappropriately long treatment period (Knight, 2019). The main impact of this project is to knockout genes that are resistant to a highly effective drug, Streptomycin, which would increase the treatment success rate and shorten the length period of treatment. This will benefit MMU through high quality publications and the potential for receiving funds to support future internal grant applications. References Ensure these are consistently formatted using MMU Harvard. Use Endnote. The library has support for referencing correctly, and using endnote: https://www.mmu.ac.uk/library/referencing Abel, D., 2019. Approved research. [online] Ukbiobank.ac.uk. Available at: [Accessed 27 October 2021]. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P., 2002. Molecular biology of the cell. 4th ed. New York, p.Isolating Cells and Growing Them in Culture. Applied Biological Materials, 2021. How to perform a Bacterial CRISPR Cas9 Knockout Experiment. [video] Available at: [Accessed 28 October 2021].

Atcc.org. 2021. Genomic DNA from Mycobacterium tuberculosis strain X004439 | ATCC. [online] Available at: [Accessed 27 October 2021]. Choudhary, E., Thakur, P., Pareek, M. and Agarwal, N., 2015. Gene silencing by CRISPR interference in mycobacteria. Nature Communications, [online] 6(1). Available at: [Accessed 27 October 2021]. Cohen, K., Stott, K., Munsamy, V., Manson, A., Earl, A. and Pym, A., 2020. Evidence for Expanding the Role of Streptomycin in the Management of Drug-Resistant Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy, [online] 64(9). Available at: [Accessed 27 October

2021]. Collias, D. and Beisel, C., 2021. CRISPR technologies and the search for the PAM-free nuclease. Nature Communications, [online] 12(1). Available at: [Accessed 27 October 2021]. Hille, F. and Charpentier, E., 2016. CRISPR-Cas: biology, mechanisms and relevance. Philosophical Transactions of the Royal Society B: Biological Sciences, [online] 371(1707), p.20150496. Available at: [Accessed 27 October 2021]. Knight, G., McQuaid, C., Dodd, P. and Houben, R., 2019. Global burden of latent multidrugresistant tuberculosis: trends and estimates based on mathematical modelling. The Lancet Infectious Diseases, [online] 19(8), pp.903-912. Available at:

[Accessed 27 October 2021]. Lino, C., Harper, J., Carney, J. and Timlin, J., 2018. Delivering CRISPR: a review of the challenges and approaches. Drug Delivery, [online] 25(1), pp.1234-1257. Available at: [Accessed 27 October 2021].

Liu, X., Zhou, X., Li, K., Wang, D., Ding, Y., Liu, X., Luo, J. and Fang, C., 2020. A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice. PeerJ, [online] 8, p.e8491. Available at: [Accessed 27 October 2021]. Ncbi.nlm.nih.gov. 2021. Find transcript sequences for a gene. [online] Available at: [Accessed 27 October 2021]. O’Neill, J., 2016. Tackling Drug-Resistant Infections Globally. [online] Amr-review.org. Available at: [Accessed 27 October 2021]. Ncbi.nlm.nih.gov. n.d. Polymerase Chain Reaction (PCR). [online] Available at: [Accessed 27 October 2021].

Orders.synthego.com. 2021. CRISPRevolution sgRNA EZ Kit - Synthego Genome Engineering Solutions. [online] Available at: [Accessed 27 October 2021]. Palomino, J. and Martin, A., 2014. Drug Resistance Mechanisms in Mycobacterium tuberculosis. Antibiotics, [online] 3(3), pp.317-340. Available at: [Accessed 27 October 2021]. Qiu, P., Shandilya, H., D'Alessio, J., O'Connor, K., Durocher, J. and Gerard, G., 2004. Mutation detection using Surveyor™ nuclease. BioTechniques, [online] 36(4), pp.702-707. Available at: [Accessed 27 October 2021].

Smith, T., Wolff, K. and Nguyen, L., 2012. Molecular Biology of Drug Resistance in Mycobacterium tuberculosis. Current Topics in Microbiology and Immunology, [online] pp.53-80. Available at: [Accessed 27 October 2021]. Synthego.com. 2021. Synthego | Full Stack Genome Engineering. [online] Available at: [Accessed 27 October 2021]. Tuladhar, R., Yeu, Y., Tyler Piazza, J., Tan, Z., Rene Clemenceau, J., Wu, X., Barrett, Q., Herbert, J., Mathews, D., Kim, J., Hyun Hwang, T. and Lum, L., 2019. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nature Communications, [online] 10(1). Available at: [Accessed 27 October 2021]. Yang, W., Yoon, A., Lee, S., Kim, S., Han, J. and Chung, J., 2017. Next-generation sequencing enables the discovery of more diverse positive clones from a phage-displayed antibody library. Experimental & Molecular Medicine, [online] 49(3), pp.e308-e308. Available at: [Accessed 27 October 2021]....