CRISPRs: An Exploration into Spacers and Captured Viral Similarity By: Katie Richeson November 19, 2009 Genomics Laboratory Methods Davidson College, Davidson, NC

Basic Viral Background Terminal Repeats: Insertion Excision (inverted or direct repeats)

Basic CRISPR Background CRISPR= Clustered Regularly Interspaced Short Palindromic Repeats

Proposed Function of CRISPRs

Two Main Questions 1.How many and what viruses are captured in our species’ spacers? 1.Are the viruses captured in our species’ spacers in other species’ genomes and/or spacers?

Question One: Viruses in Our Species’ Spacers 3 Viruses in 77 Spacers – Archaeal BJ1 Virus (E-value= 0.005) Myriad of halophiles, archaea, various types of fungi, ticks, chicken, salmon, cows – Burkholderia phage (E-value=0.02) Various types of bacteria and a few flowering plants – Mycobacterium phage (E-value= 0.31) Different types of bacteria and fungi

Small Fraction of Viral Genomes Available Entrez Genomes: 2362 whole viral genomes available Estimated Number of Viruses in the World – More than 1 billion microbes – “The estimated number of DNA viral species has been reported to be as low as 1440 in hot springs, and as high as 129,000 in the open ocean” (Willner et al., 2009) The best estimate is that we have access to viral sequences for about 2% of the viruses worldwide

Question Two: Are the viruses captured in our species’ spacers in other species’ genomes and/or spacers? 1.Determine what other species to explore 1.Determine if these viruses are in these other species’ genomes 1.Determine if these viruses are captured in the spacers of these other species – Find CRISPRs and spacers for each species

Question Two: Do these viruses exist in other species’ genomes or spacers?

Question Two: Do footprints of these viruses exist in other species’ genomes? All 4 species have significant hits when blasted against these 3 viruses

Question Two: Were any of these 3 viruses captured in the spacers of these 4 other species? Determine CRISPRs and spacers... – Halorhabdus utahensis and Haloarcula sinaiiensis have confirmed CRISPRs as identified by CRISPRFinder – Haloarcula vallismortis and Halobacterium salinarium R1 have no confirmed CRISPRs, but appear to have CAPs

Question Two: Were any of these 3 viruses captured in the spacers of these 4 other species? None of these viruses were captured in the spacers of these other 4 species... Yet footprints for all 3 of these viruses exist in all 4 species

Conclusions Our species has 3 identifiable viral snippets captured in spacers Although these 3 viruses have infected the 4 other species, they have not been captured by their spacers – Species’ cannot be related based upon viral footprints in their genomes because viruses leave footprints in many other organisms Exploring Archaeal BJ1 virus, I found that none of the 8 species in consideration had captured this virus in any of their spacers – It is difficult and inaccurate to relate species’ based upon spacers capturing similar viruses