The Mystery of CRISPR-Cas

     The CRISPR-Cas system has been a recent subject of study for the previous twenty years, with a increased interest in the past few years for biomedical applications utilizing the Class II system, which exploits a relatively simple process to enact DNA or RNA cleavage of desired targets. The CRISPR system relies on an array of repeats and spacers to identify previous viral threats, the spacers primarily being segments of foreign genetic material stored in between highly conserved repeat sequences created by the cell. The Class II system, in general, universally involves one enzyme that is responsible for recognizing three to five base pair PAM sequences and cutting foreign targets. The primary protein of interest thus far has been Cas-9, which can cut double stranded DNA with a very high efficiency and specificity, but other similar Cas proteins exist. Alternatively, the Class I system has been understudied due to the complexity of the system in comparison to the Class II category of CRISPR-Cas because it usually involves a multiprotein complex made up of a combination of cmr, csm, and cas genes in order to recognize and degrade foreign DNA and RNA. This system relies on the use of this complex, and a potential duality of protein functions. Proteins having more than one function seems to be necessary for the suspected CRISPR-Cas system in  Deinococcus aquaticus to be operational, since it does not have very numerous CRISPR or csm proteins. Bioinformatic analysis indicates five separate array regions, with one region geographically located directly upstream of the identified proteins, being cas1, cas2, cas6, and csm2, csm3, and csm5. Ideally, we would like to be able to confirm that D. aquaticus PB 314 does in fact have a functioning system, capable of recognizing spacer sequences, carrying out cutting events, and potentially being able to integrate new spacers. Since this species has not been confirmed transformable as of yet, the current goal will be to provide a proof of concept for future experiments, as well as collect novel CRISPR data for insight on baseline cas gene expression in a cell in comparison to stimuli to upregulate specific cas proteins. 

    Deinococcus geothermalis has been shown to be readily transformed by early versions of the plasmid, Prad1, which may be useful in an assay with qPCR to evaluate gene expression of cas proteins. D. geothermalis also happens to have one of the most detailed arrays known out of the Deinoccocus genus, which is extremely limited. We are hoping to create a protocol that will estimate the timing of the CRISPR system and accurately evaluate a change in gene expression, potentially over a period of time anywhere from one to forty-eight hours. In order to have a chance to do so, we must first successfully transform D. geothermalis with Prad1 or one of its ancestor plasmids, which may be genetically engineered later on with either a known spacer sequences from its own array to instigate  a cutting event. Once this is accomplished, the plasmid will be engineered with a spacer sequence to hopefully observe a difference in transformation efficiency between cells that have been transformed with a spacer sequences versus a plasmid with a nonsense sequences in place of the spacer. Both of these plasmids would have markers, such as antibiotic resistance, to select for and visually represent transformation efficiency. In this way, a low transformation efficiency of the plasmid containing the spacer compared to a standard plasmid seen on a plate with antibiotics may indicate that the plasmid is being destroyed by CRISPR genes and linearizing the plasmid so that the antibiotic resistance gene cannot be expressed. 

D. aquaticus proposed CRISPR-Cas regions that are of interest to test activity in future projects, once protocol can be developed with D. geothermalis


    There is no current research that has been found that has done qPCR for standard CRISPR function, but that is likely due to the lack of biomedical applications in doing this work. That being said, the CRISPR systems currently known are still very much a mystery in how they operate, how their arrays are maintained, and how systems have been shared and inherited between species since there is not a high level of conservation even within a single genus. By starting with preliminary steps next week, we may be able to add the story and use this development of protocol for future species and isolates that will be sequenced. 


Sources: 

Liu, T., Pan, S., Li, Y., Peng, N., & She, Q. (2017). Type III CRISPR-CAS system: Introduction and its application for genetic manipulations. The CRISPR/Cas System: Emerging Technology and Application. https://doi.org/10.21775/9781910190630.01

Yoshimi, K., & Mashimo, T. (2022). Genome editing technology and applications with the type I CRISPR system. Gene and Genome Editing, 3-4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013

You, L., Ma, J., Wang, J., Artamonova, D., Wang, M., Liu, L., Xiang, H., Severinov, K., Zhang, X., & Wang, Y. (2019). Structure studies of the CRISPR-CSM complex reveal mechanism of co-transcriptional interference. Cell, 176(1-2). https://doi.org/10.1016/j.cell.2018.10.052

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