Elimination of Antibiotic Resistance Genes Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Systems Mitesh Agrawal,

Biomedical Engineering Advisors: Dr. Eric Gaucher, Dr. Mark Styczynski, Dr. Joshua Weitz I. Overview Overuse of antibiotics in medical settings has created the problem of antibiotic resistant bacteria, popularly referred to as super bugs. These super bugs are created when a microorganism survives treatment with an antibiotic, and then spreads this immunity throughout the population through horizontal gene transfer and natural selection. The infections caused by these bacteria are difficult to treat, and in some cases even life threatening. One of the most well-known common examples of a super bug often is Methicillin-resistant Staphylococcus aureus, or MRSA. Serious, invasive MRSA infections occur in approximately 94,000 people and are associated with approximately 19,000 deaths each year [1]. Antibiotic resistance in bacteria is often located on a plasmid, a circular piece of DNA separate from chromosomal DNA, for ease of horizontal gene transfer among microorganisms. The experiment outlined in this proposal aims to engineer a unique method of reverse vaccination in which an antibiotic resistant plasmid is specifically targeted and inactivated, rendering the bacteria vulnerable to antibiotics. The novel method we are developing utilizes a CRISPR system (Clustered Regularly Interspaced Short Palindromic Repeats) contained on a separate plasmid to inactivate the foreign DNA [2]. The CRISPR system is a type of immunity used by about 40% of Bacteria and about 90% of Archaea [3]. It consists of a series of proteins, called cas proteins, which are associated with a series of spacers separated by short palindromic sequences. These are used by the cell to recognize foreign DNA (either plasmid or viral) and target it for deactivation. If the DNA is not recognized and the cell survives the foreign plasmid or phage, it then saves pieces of the foreign DNA in subsequent spacers, to be used in future recognition of an attack. (Figure 1) Using a modified CRISPR system that is delivered via a viral vector, our research aims to demonstrate that antibiotic resistance genes can be targeted with the CRISPR system, rendering the bacteria more susceptible to treatment. More specifically, artificial kanamycin resistance in a non-pathogenic E. coli strain will be targeted, which will serve as a safe proof of concept toward a possible super bug treatment sometime in the future. II. Objectives and Techniques Production of a Functional CRISPR Plasmid The first step in the project is to create a functional CRISPR system on a plasmid compatible with E. coli as described by Pougach et al [4]. Plasmid transformation will be verified with a selectable marker and fluorescent tag to confirm expression. The next step in the process is to create a spacer corresponding to kanamycin resistance. This will be achieved in one of two ways: either via natural activity of the CRISPR system on a plasmid in cells exposed to a kanamycin resistance plasmid, or via the process developed by Barrangou et al [3] to encode the spacers. Introduction of CRISPR Plasmid into a Kanamycin Resistant E. Coli Strain Kanamycin resistance will be induced in a non-pathogenic strain of E. coli by introduction of a plasmid with encoded kanamycin resistance. The antibiotic resistance targeting CRISPR plasmid will then be introduced into this transformed E. coli strain. To verify that CRISPR is present and expressed, a fluorescence tag will be engineered into the CRISPR plasmid, and confirmed by fluorescence microscopy. The E. Coli strain with kanamycin resistance plasmid and no CRISPR plasmid will be plated on kanamycin plates and their growth

should be observed. Some of these resistant colonies will be selected to introduce the CRISPR plasmid. To verify that the kanamycin resistance gene has been inactivated, colonies with CRISPR plasmid will be replica plated on kanamycin plates and non-selective plates. If successful, no colonies should be expected on kanamycin plates whereas all colonies should be observed on non-selective plates.

Figure 1: Diagram of the mechanism of CRISPR[5] III. Research Background/ Team Setting This project has been undertaken by the Georgia Tech iGEM 2011 team that will compete at the iGEM Jamboree annually held at MIT. iGEM which stands for International Genetically Engineered Machines is the premiere undergraduate synthetic biology competition where 130 teams from all over the world compete annually. I was a member of the silver medalwinning first GT iGEM team in 2010 and have continued on to the GT iGEM 2011 team. I have been also working under the mentorship of Dr. Todd McDevitt at McDevitt Stem Cell Laboratories for the past two semesters. It is hoped that with this research, new ways for targeting antibiotic resistance will be realized, helping to spark further research and innovation in real world applications of CRISPR systems IV. References [1]Klevens R., Morrison M., et al. (2007), JAMA 298(15): 1763-1771. [2]Babu, M., N. Beloglazova, et al. (2011). Molecular Microbiology 79(2): 484-502. [3]Barrangou, R., C. Fremaux, et al. (2007). [4]Pougach, K., E. Semenova, et al. (2010), Molecular Microbiology 77(6): 1367-1379. [5]Horvath P., Barrangou R.,(2010), Science, 327(5962), 167-170.