The Use of Type III-A CRISPR-Cas Systems to Determine Sequence Importance in E. coli’s Defense Against T4 Phage

• Hannah Weile, Biological Sciences, University of Delaware

• Jeremy Bird, Department of Biological Sciences, University of Delaware

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a defense mechanism that protects bacteria from foreign invaders, such as viruses called bacteriophages. In 2009, this technology was first utilized as a genome editing tool, which can accelerate disease defense research as bacteria are becoming antibiotic-resistant. However, research describing CRISPR’s use for bacterial genome editing rather than human genome editing is sparse. This study aims to close this gap in molecular biology research, specifically within the model of E. coli bacteria and one of its seven commonly infecting bacteriophages, T4 phage. The question focused on throughout this research asks when adding Type III-A CRISPR system in E. coli to protect against invading T4 phage RNA that targets the genes for either the phage’s DNA polymerase or sigma factor, which of the two CRISPR inserts will offer better protection? Before working with E. coli and T4 phage, the efficacy of the methodology was tested with a less invasive phage, Andhra, its corresponding bacteria, S. epidermidis, which uses a CRISPR Type III-A system. For my project, I am cloning T4 phage gene targeting sequences into a Type III-A CRISPR system expressing plasmid. The final plasmids will be used in a phage plaque assay to determine the number of plaque-forming units (PFUs). The hope is to see a significant decrease of plaques with the expression of the CRISPR defense system. This research will allow for a better understanding of the T4 phage genome and the potential utilization of Type III-A CRISPR systems as a tool in E. coli. In the future, this protocol will be used to test more of the 288 genes in T4 phage to see whether CRISPR systems using other gene inserts are better or worse for bacterial protection.