My name is Jarlath Nally, and I'm part of the research team here at the National Animal Disease Center in Ames, Iowa, and we work on leptospirosis. Leptospirosis is a neglected disease. It's a global disease.
And really, we have a very limited understanding of how these unique group of bacteria cause infection. So today's presentation is about providing an overview of a new tool that's available to facilitate the evaluation of pathogenic mechanisms of leptospirosis by specifically silencing specific genes expressed by leptospira, using a technique called CRISPR interference. However, the ability to select newton colonies is very dependent on providing the best growth media for these fastidious leptospiras.
So, first of all, we're going to introduce you to Rick Hornsby, who was very briefly introduce a new media to propagate these fastidious bacteria. And then we'll introduce you to Louis Fernandez, who will talk about the CRISPR interference methodology to silence target genes within leptospira. Hi, I'm Rick Hornsby.
Since their identification by Anada over 100 years ago, leptospiras have typically been isolated and propagated at 29 degrees Celsius, a temperature that reflects neither the environment nor the mammalian host body temperature. 10 years ago, USDA ERS began researching improvements on leptospira media and out of this research, Hornsby-Alt-Nally media was developed. Using this media, clinicians and researchers have been able to more rapidly isolate and propagate leptospiras from field cases and in in vitro research at a temperature and in an environment that more closely emulates the mammalian host.
We continue to optimize this media, but as it stands, HAN has demonstrated potential to significantly improve veterinary and human clinical isolations in vitro research and the development of more efficacious vaccines for the prevention of leptospirosis. Hi, I'm Louis Fernandez. So far, the pathogenesis of leptospirosis remained under-explored, mainly due to the lack of effective and easy to perform genetic tubes.
Here we're gonna describe a new technique called CRISPR interference or CRISPRi. And this technique is very simple because we only need to express two components, one DNA binding protein called dead Cas9, or dCas9, and a chimeric RNA molecule called single-guide RNA. And the complex dCas9 and guide RNA are gonna make base pairing to our target gene and therefore reduce or even block transcription.
Also by using the newly described HAN media, we could drastically decrease colony formation time for a mutant recovery. The first step is defining the protospacer that will be contained in the guide RNA. This sequence comprises 20 nucleotides that will define the base pairing to the desired gene targets.
Submit a nucleotide sequence to the web server CHOPCHOP, with parameters defined for streptococcus pyogenes, Cas9, and protospacer adjacent motif, NGG. Based on the results, select protospacers with the best scores within the minus trend. The NGG motif will not be included in the final sequence.
We have been using the lipL32 promoter for expressing the single-guide RNA, which will contain the variable 20 nucleotides at five-prime at a conserved dCas9 scaffold. After obtaining the cassette it will be relegated into the PMaOri. dCas9 plasmid at the Xma1 restriction site.
For conjugation leptospira cells are grown at 29 or 37 degrees celsius in HAN media. One day before a conjugation, donor equalized cells are grown in LB media supplemented with diaminopimelic acid, DAP, and spectinomycin. Next, in the day of conjugation grill the saturated E.coli cultures in LB plus DAP.
Inside a bio safety hood, assembled the filtration apparatus by placing the membrane filter on top of the glass base, followed by 15 ML glass funnel. Both pieces are held together by this spring clamp. Then connect the glass to a vacuum pump, add five ML of leptospira cultures to the funnel.
Next, add the E.Coli beta 2163 strain containing the plasmid of interest. The volume will vary according to the optical density of the culture. We are aiming to one-to-one cell pipersion.
Turn the vacuum pump, now liquids we will get through the membrane and cells will be concentrated on top of it. Take the filter and place it on to EMJH plate plus DAP, bicurier side up. Incubate the plates at 29 degrees celsius for 24 hours.
Then recover the filters from the plates and place it into 50 ML conical tube. Use one ML of liquid HAN media to recover the cells from the filter by pipetting. Visualize the recovered cells by dark field microscopy.
At this stage equivalent numbers of E.coli and leptospiras can be seen. 100 to 200 microliters are used to spread the bacteria onto HAN plates containing spectinomycin. At this stage, DAP is omitted and E.coli will not grow.
Plates are incubated at 37 degrees celsius and 3%CO2. Colonies should be apparent between eight to 10 days. Leptospira interrogans colonies are a little tricky to visualize.
Here we are showing overgrown colonies to make it easier to see them. Add 100 microliters of liquid HAN, to 1.5 ML tubes for recovering the mutants. It is recommended to take at least three colonies from each plate.
Using a pipette tip, individual colonies will be retrieved from the plates. Agger is expected to be taken alone. Colonies should be taken from control plates containing MTP PMaOri.
dCas9 plasmid. And plates with leptospiras containing plasmid with both dCas9 and guide RNA designed for the target gene. The recovered cells can now be visualized under dark field microscopy to confirm the presence of leave viable leptospiras.
After visualization of leave leptospiras, transfer cells to liquid HAN media containing spectinomycin. After culture growth, cells can be now recovered for silencing confirmation and phenotype evaluation. If antibodies against the target proteins are available immunoblot is a straight forward methodology to evaluate silencing.
Leptospiras containing pMAOri. dCas9 alone, or with the guide RNA Cas are evaluated by PCR, wood flanking primers. DMT plasmid will result in a band of approximately 300 base pair and an only kind of around 700 is attained when the guide RNA cassette is present.
Immunoblots using whole cell extracts are performed for silencing confirmation, lipL32 protein expression is abolished in cells containing plasmids expressed in dCas9 and guide RNA targeting this gene. And both link a and link B are absent in cells containing the guide RNA, is specific for both genes. The control lipL41 can be seen in all lanes.
So we've used CRISPR interference on leptospira interrogans, we've also used it on several additional pathogenic species of leptospira. We've used it to silence specific genes, as well as silencing the expression of small non-coding RNAs. Our research interest is in understanding pathogenic mechanisms of infection, so we typically look to identify variance factors, but obviously you can silence your own gene of choice and according to your own area of research.
And for example, that might be motility or understanding how leptospiras persist in the environment, or metabolism. In any case, you should be able to generate a specific mutant that will allow you to perform functional genomics and understand the biological significance of the gene in question. So from all of us here at the National Animal Disease Center, we wish you the very best of luck with your research.
