Anti-CRISPRs and CRISPR-Cas: Characterization and Biotechnology

Abstract Bacteria and phages (bacterial specific viruses) have been undergoing an evolutionary arms race for billions of years, whereby bacteria evolve mechanisms to inhibit phage infection and phages evolve mechanisms to evade bacterial defenses. Consequently, there are several mechanisms used by bacteria to inhibit phage infection. These include inhibiting phage adsorption, restriction modification systems, and the more recently discovered CRISPR-Cas immune systems. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci together with their accompanying CRISPR-associated (Cas) genes form the only known bacterial adaptive defense mechanism that effectively protects against the transfer of mobile genetic elements (MGEs) such as bacteriophages. CRISPR-Cas systems use an RNA-guided nuclease to bind and cleave foreign DNA, presenting a powerful barrier to phage infection. This strong evolutionary barrier led phages to evolve small protein inhibitors of CRISPR-Cas called anti-CRISPRs. In the first section of my work, I characterize the structure, function, and mechanism of action for an anti-CRISPR that inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa strain 4386. I show that beyond simply inhibiting the CRISPR-Cas system, anti-CRISPR AcrIE2 converts the CRISPR-Cas system from a DNA degradation complex to a transcriptional regulator. This modification suggests that anti-CRISPR proteins may function to do more than simply inhibit CRISPR-Cas targeting. Although CRISPR-Cas systems are a manifestation of the evolutionary arms race between bacteria and phages, CRISPR-Cas systems have also been used for genome editing in various organisms for research purposes. Considering this previous work, I developed a type I-E and type II-A CRISPR-Cas genome editing system to manipulate the genome of different P. aeruginosa strains for research. In the second section of my work, I discuss the methods I developed to efficiently edit the genome of P. aeruginosa. My work on genome editing in P. aeruginosa has and will allow for the development of new P. aeruginosa mutants for research purposes. Collectively, my work provides insight into the evolutionary interactions between phages and bacteria in the context of CRISPR-Cas and anti-CRISPRs. Moreover, it provides a genome editing tool for future P. aeruginosa studies.

Department dc:contributor.department

Biochemistry

Year dc:date.issued

2019

Author dc:creator

• Mejdani, Marios

Advisor dc:contributor.advisor

• Davidson, Alan R

• Anti-CRISPR
• Bacteriophages
• CRISPR-Cas
• Gene regulation
• Genome editing

Handle dc:identifier.uri

http://hdl.handle.net/1807/102773

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/102773