We no longer have to imagine the disruptive, destabilizing and deadly effects of widespread respiratory biothreats; the Covid-19 pandemic has clearly demonstrated the global security threat created by aerosol transmission of disease. While the origin story of Covid-19 did not begin in US or Soviet biological laboratories, that of Francisella tularensis did. Francisella tularensis (pictured below) is considered a Category A critical biological agent as it can easily be disseminated through aerosol and is the causative agent of the deadly disease tularemia. It requires inhalation of only 25 microscopic cells to cause disease in humans. Its non contagious and deadly nature makes it useful as a bioweapon because it will only infect the targeted population.
Microscopic image of F. tularensis. Source: National Institute of Allergy and Infectious Disease (NIAID) Laboratory of Intracellular Parasites, Tularemia Pathogenesis Section.
Francisella novicida being released from a lysed macrophage. SEM image provided by M. Neal Guntzel and Annette R. Rogriguez.
Studying highly infectious disease causing bacteria is challenging due to high safety precautions. To mitigate the risks of infection or unintended outbreak while researching microbes, scientists have developed Biosafety Levels (BSL), outlining safety precautions that align with the level of riskiness for each bacteria (See image). As you can imagine, research with lower BSL requirements can occur in more labs with a lower risk while high BSL levels can only occur in laboratories equipped to handle and mitigate high risk bacteria.
Drawing by Kathryn Murphy depicting safety precautions from BSL1 to BSL4. Both personal protective equipment, lab equipment, and structural facility safety requirements increase with heightened BSL levels. The heightened safety measures correlate with increased riskiness of bacterial research determined by transmissibility and severity of infection.
Bacterial surrogates are highly similar to their pathogen causing cousins but require a lower safety precaution. As such, many labs opt to research bacterial surrogates to understand broad information about highly pathogenic bacteria. Novicida's benign nature in humans led to its use as a bacterial surrogate for studying tularensis in order to better understand molecular mechanisms of disease causing tularensis. In addition to a high degree of nucleotide similarities, novicida induces disease in mice and can infect macrophages (Bachert et al.). Furthermore, novicida can be handled under more relaxed BSL-2 safety precautions compared to the stricter BSL-3 precautions required for research utilizing tularensis. The CDC also requires Sars-CoV2 cultures to be handled under BSL-3 precautions. Altogether, this makes novicida an ideal bacterial surrogate; comprehensively researching Francisella novicida can help the scientific community better understand the more dangerous tularensis and work towards developing robust treatment options to mitigate its threat in an attack.
One aspect of Francisella novicida that contributes both to its virulence as well as its defense against invaders is CRISPR-Cas systems. While CRISPR-Cas systems are not native to Francisella tularensis, research in bacterial immune systems of CRISPR-Cas contributes to overall understanding of bacterial virulence, bacterial immune defense, and may point to analogous mechanisms in tularensis.
CRISPR-Cas is most widely known for its genome editing capabilities, however it originated as an adaptive immune system in prokaryotic bacteria. When infection occurs in bacteria, CRISPR arrays genetically record sequences of nucleic acids from the invading bacteriophage or plasmids, essentially creating a guest log of unwelcome invaders. This information is held on the CRISPR protospacer sequence which forms CRISPR arrays in combination with repetitive sequences (Ratner and Weiss). To prevent another unwelcomed visit by the bacteriophage or plasmid, the CRISPR array makes copies of this guest log, in the form of CRISPR RNA transcription. CRISPR arrays may contain numerous spacers interspersed with repeats. In order to mobilize these invader recognizing sequences, the arrays are processed and cut so that only one spacer and the flanking repeat sequence remains. Individual CRISPR RNAs (crRNAs) attach to Cas proteins forming the CRISPR-Cas system. When crRNAs' spacer sequence recognizes and binds complementarily to the invading sequence, the Cas protein will destroy the foreign DNA. There are numerous CRISPR-Cas systems each with different or multiple proteins driving this process. Class I CRISPR-Cas systems require multiple proteins to target the invader DNA and cleave it. Class II systems are simplistic in their design, requiring only one protein to drive the recognition and destruction of unwelcome guests and therefore have been the focus for genomic engineering. Cas9 is arguably the most well known Cas protein, while scientific research has expanded to Cas12a and other Cas proteins.
Artwork by Kathryn Murphy depicting how bacterial cells utilize CRISPR immunity to fight off recurring viral infections.
Francisella novicida utilizes both CRISPR-Cas12a (FnoCas12a) and CRISPR-Cas9 (FnoCas9) in its adaptive immune system, both believed to influence bacterial virulence and transformation inhibition. A recent study published by Ratner and Weiss in 2020 explored CRISPR-Cas9 and CRISPR-Cas12, native to Francisella novicida to explore the function of both systems. Prior to this study, examination of FnoCas12a virulence and the role of FnoCas9 and FnoCas12a in DNA defense had not been studied. They determined multiple CRISPR-Cas systems can coexist in a bacteria, working in concert with each other to provide robust immunological defenses. Additionally, they wondered whether Cas9 and Cas12a have similar efficiency for targeting of foreign DNA and whether they were effective in the presence of each other. They found both Cas9 and Cas12a could target the engineered artificial target, both individually, and in combination with the other.
Once the authors proved that Cas9 and Cas12a function independently of each other to recognize and defend against unwelcome invader DNA, they explored the efficiency of each CRISPR-Cas system. To do so, they introduced a new spacer which contains the genetic information of a target "engineered invader" into the F. novicida's CRISPR array (Figure 5A). This is indicated by the black box labeled "new spacer." When the CRISPR array is divided into individual crRNAs, combined with Cas proteins, and replicated, it should recognize the engineered target sequences and destroy them. To test the efficiency of the reprogrammed Cas12a and Cas9, they then created a target plasmid and added PAM sequences. PAM sequences are sequences of nucleotides directly upstream or downstream from the DNA target and ensures CRISPR-Cas systems are attacking unwelcome invader sequences rather than themselves. Because Cas proteins in complex with the crRNAs lack PAM sequences, the CRISPR-Cas system will not unintentionally target themselves. The upstream location of Cas9 and downstream location of Cas12a (red) PAM sequences with respect to the engineered DNA target on the plasmid is illustrated in Figure 5A. With the help of both PAM sequences, reprogrammed Cas12a and Cas9 should be able to properly differentiate and target the sequence.
Reprogrammed Cas12a and reprogrammed Cas9 efficiently targeted the non-native "engineered invader" sequence, resulting in a significant difference in % escaped colonies between Cas12a and Cas9 target compared to the control (Figure 5B). This indicates that Cas9 and Cas12a systems can successfully and with similar efficiency, target transformed plasmids. Additionally, when both reprogrammed Cas9 and Cas12a were simultaneously introduced to target the nonnative invader, they restricted transformation at similar efficiencies as individually. Not only did this study show that Cas12a and Cas9 CRISPR systems can be reprogrammed to identify new target sequences, it also indicates that they efficiently work independently of each other. As such, one could utilize both systems to provide a multi-prong targeting strategy against invading nucleic acid hazards (Ratner and Weiss). CRISPR-Cas systems innate to many bacterial species contribute to bacterial immune systems. Understanding bacterial immunity in Francisella novicida deepens our understanding of CRISPR technology and potential applications in humans and bacteria.
Bacterial surrogates remain ever important as Ratner and Weiss unlocked our understanding of dual-defense CRISPR-Cas immune systems native to novicida. Learning mechanisms of bacterial immunity can inform scientists as they try targeting and evading the defense system to prevent bacterial infections in humans. This study opens the door for future research into usage and applications of CRISPR-Cas gene editing.
No comments:
Post a Comment