Investigating CRISPR Spacer Sequences of Escherichia coli in NJ/NY Metropolitan Area

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) is a DNA sequence, found in bacteria, that functions as protective system against bacteriophages. CRISPR DNA sequence is characterized by the presence of short, identical nucleotide repeats that are interspaced by spacers. The spacer sequences are unique in that they are records of viral infections experienced by the bacterium and act as an “immunization card”. If the same virus attempts to invade again, the spacer sequence recognizes it via the help of CRISPR‐RNA (crRNA) and CRISPR‐associated (Cas) proteins. Subsequently, Cas proteins cut the viral DNA to eliminate the threat. Thus, CRISPR/Cas system provides an effective mechanism for bacteria, such as Escherichia coli (E. coli), to resist infections from bacteriophage, but it could also pose challenges to phage therapy, which utilizes bacteriophages to kill bacteria. The focus of this research is to examine spacer sequences from soil samples collected around NY/NJ metropolitan area at various timepoints, locations, and weather conditions. To accomplish this goal, the genomic DNA was extracted from E. coli bacteria from the soil and purified. The CRISPR locus was amplified and purified using polymerase chain reaction (PCR) and gel electrophoresis, respectively. The sequences were run through the CRISPR Finder program to identify spacer sequences, and NCBI’s Blast alignment was carried out to determine the specific E. coli strain associated with the sequence. We have identified several new spacer sequences, which could putatively link the history of bacteriophage infections of the E. coli bacteria to the unique environments the bacteria are located.Support or Funding InformationWe would like to thank the Chemistry Department at NJCU. This research was funded by National Institutes of Health (NIH), grant number R21DA046223.