In Dr. Shariat’s lab (aka the “CRISPR Crew”), we’re all rising sophomores and we have three main research focuses: the function of the CRISPR-Cas system, the applications of the CRISPRs for sub-typing, and the use of the phage therapy in controlling plant pathogens. We study two closely related bacterial pathogens: Salmonella enterica and Erwinia amylovora.
Clustered Regularly Interspaced Short Palindromic Repeats with their associated cas genes (CRISPR-Cas) are found in 48% of sequenced bacterial genomes. CRISPR-Cas systems are typically used as a bacterial defense mechanisms against foreign DNA such as bacteriophage and plasmids.
CRISPR-Cas systems contain cas genes, typically located next to a CRISPR-Cas array. These arrays consist of spacers and direct repeats, which code for RNA that guide a cas nuclease to foreign DNA injected by phage, thus targeting the foreign DNA for destruction. The spacer sequences are generally derived from foreign DNA elements. CRISPR-Cas systems are adaptive: as the bacteria comes into contact with the new phage, spacers can be added to the array that are specific to that particular phage.
Function, Function, Function!
Jake and Kaelea: Most studied Salmonella genomes have been shown to contain a CRISPR-Cas system, with eight cas genes and two arrays, CRISPR 1 and CRISPR 2. CRISPR 1 is adjacent to the cas operon. Evidence suggests that the Salmonella CRISPR-Cas system is not used for immunity, as there is a lack of spacer acquisition and that only a few spacers actually match that of phage sequences. Interestingly, Salmonella has maintained the genetic integrity of its CRISPR-Cas system as seen by the lack of mutations within the cas genes and spacers. We spend much of our time in lab growing different types of Salmonella and performing assays on components of the CRISPR-Cas system. Jake can now do bacterial RNA preps in his sleep!
Celine: When we can drag her away from Phage work, Celine is looking for novel CRISPR-Cas systems in other bacteria.
Using CRISPRs as Molecular Fingerprints
Dorothy and Stefani:
Dorothy and Stefani are using a subtyping technique called CRISPR-MVLST to identify specific strains of Salmonella found in birds. CRISPR-MVLST uses sequence information from both CRISPR1 and 2 plus two virulence genes. Spacer differences in the CRISPR arrays and single nucleotide polymorphisms (SNPs) in the virulence genes are used to define and separate individual Salmonella strains.
For analyzing the CRISPR arrays, we use a macro, which only displays the spacer sequences (the bits of DNA that come from phage and which are unique). The macro generates an image like the one to the left where different colored boxes represent different sequences.
Dorothy is investigating a particular type of Salmonella that is often found in chickens and occasionally in humans. She’s been looking at human isolates of Salmonella and is finding some cool CRISPR patterns!
Stefani is a rising senior at Biglerville High School and is working in our lab as part of new program “Bridging the Furrow Ag-Research Training” for URM high school students. She’s been looking at strain differences in Salmonella isolated from different songbirds from different states, including the American Goldfinch and the Brown Headed Cowbird.
Cameron’s project focuses on the use of the Salmonella CRISPR array as a means of identifying what strains are present in a complex sample of Salmonella strains. Each strain has a slightly different composition of known unique spacers. By identifying these spacers, they are able to identify different Salmonella strains. Last week we traveled to to collect samples at a poultry facility: many chickens and lots of chicken poop. Check out our cool suits though!
And Cameron found lots of Salmonella – he’s doing PCR analysis of the CRISPRs as we write!
Celine and Caleb:
Bacteriophage can be found anywhere that bacteria are found and have been studied for over 100 years. They are a specific type of virus that infect and kill bacteria and as such, can provide an alternative therapy to antibiotics in treating bacterial infections. Celine and Caleb have been isolating and purifying phage against Erwinina amylovora, a bacterial pathogen that infects apple trees.
Collecting Fire Blight Samples
Here’s some local trivia for you: did you know that Pennsylvania is the fourth largest producer of apples in the United States? And that 75% of Pennsylvanian apples are grown right here in Adams County? Many apple orchards are within a 15-20 minute drive of campus – take a drive and see them, they’re beautiful!
In the lab, Celine and Caleb perform different assays to purify phage, including a plaque assay as shown below. They’re getting really good at this – check out all those plates!