The use of CRISPR sequences to investigate the prokaryotic adaptive immune system and its targets
Bacteria and archaea are in a constant arms race against viruses and mobile genetic elements (MGEs). Recently the clustered regularly interspaced short palindromic repeats (CRISPR) system was discovered to function as an adaptive immune system through sequence level recognition of foreign nucleotide sequences. These CRISPR arrays consist of spacer and repeat sequences, the spacers are the sequences that were incorporated from foreign DNA. The repeats separate the spacers and facilitate interactions with CRISPR associated (Cas) proteins. The Cas proteins facilitate incorporation of new sequences and degradation of target sequences. These sequences that become the spacers are called protospacers. A current topic of interest is the inclusion or adaptation of foreign sequences into the CRISPR arrays. This has led to the discovery of primed adaptation where some CRISPR types are able to adapt more rapidly to sequences that have similarity with sequences already in CRISPR arrays. In this research, the analysis of genomes with single CRISPR-Cas systems led to the identification of genomes with only one CRISPR-Cas system, specifically “I-E only” and “I-F only”. These datasets were used in combination with bacteriophage and plasmid databases to determine if the spacers’ targets clustered in foreign DNA. It was shown that for “I-F only” protospacers from bacteriophage origin showed clustering that is consistent with primed adaptation. Clustering was not found in the “I-E only” systems. This agrees with other experimental evidence suggesting I-E primed acquisition has no such bias. This study of protospacers has extended what wet experiments studies determined and indicates it is not specific to the few species studied. This research also focused on development of a bioinformatic methodology to identify sequences from viruses using genomic CRISPR arrays. It aimed to identity viruses that would infect ruminant methanogens by computational means. The experiments performed using methanogen CRISPR arrays found protospacers from viromes and a metagenome. The identification of these protospacers led to the production of three contigs. Two contigs were established from a virome and are potentially fragments of viruses that targets ruminant methanogens. One sequence was identified from a metagenome, which means it could be a fragment of a virus or MGEs that target ruminant methanogens. This shows that the methods established has the ability to identify novel viruses and MGEs that are targeted by CRISPR arrays.
Advisor: Brown, Chris
Degree Name: Master of Science
Degree Discipline: Biochemsitry
Publisher: University of Otago
Keywords: CRISPR-Cas; Methanogens
Research Type: Thesis