The CRISPR/Cas system (clustered regularly interspaced short palindromic repeats/CRISPR-associated sequences) is an adaptive immune system found in archaea (extremophiles) and bacteria. Briefly, acquired CRISPR-driven immunity is based on integration of short nucleotide sequences, called spacers, generated from homologous sequences (proto-spacers) within the genomes of invading viruses, bacteriophages and plasmids. Subsequent invasions trigger expression of complementary RNAs from the host CRISPR locus (crRNAs) that guide Cas endonucleases to cleave and destroy the foreign DNA.
Repurposing of the Cas9 endonuclease to target DNA sequences in a variety of animal models, including humans, has revolutionized genome-editing applications. As we adopt the use of this system in our own bench research, I invite you to take a step back and consider the true evolutionary power of CRISPR/Cas systems. Many bacteria employ CRISPRs for both immune and (newly appreciated) non-immune functions. Other species lack the loci entirely, and this phenomenon is largely lifestyle-based. In immune contexts, the use of CRISPRs drives rapid co-evolution among viral predators. Overall, the extraordinary evolutionary history of CRISPR/Cas systems is re-shaping our understanding of bacterial and phage biology as a whole, with unexpected ties to controlling the human microbiome and improving our struggles against antibiotic-resistant infections.
THE CRISPR-PHAGE ARMS RACE
In 1973, evolutionary biologist Leigh Van Valen proposed a metaphor to describe biological processes that are characteristic of “arms races.” The so-called Red Queen Hypothesis posits that organisms must constantly adapt, evolve, and thrive against ever-evolving opposing organisms within the same environment; it was named after the line, “Now, here, you see, it takes all the running you can do, to keep in the same place!” proclaimed by The Red Queen in Through the Looking-Glass. A prime example of this presents itself in the bacterial CRISPR immune system.
Bacteria employ a diverse range of innate mechanisms to defend themselves against invading phage populations. The uniquely adaptive CRISPR/Cas defense system creates a true memory of infection, and population-level protection is maintained by rapid, individual-level diversification of spacer repertoires in bacteria. In response, bacteriophages counter-evolve in a variety of ways, mainly by mutating or deleting highly targeted areas of their genomes.
Phage co-evolution to CRISPR was first described by Deveau et al, from experiments using the lactic bacterium Streptococcus thermophilus. The authors discovered that single nucleotide mutations (SNPs) and deletions occurred at high frequencies within or near the proto-spacer of bacteriophages in response to S. thermophilus CRISPR activity. More recent experiments by Sun and colleagues tracked how co-evolving host and phage populations are established within surprisingly few generation times. They cultured S. thermophilus with a specific phage, and used deep sequencing to trace the CRISPR diversification within a population derived from a colony with phage-specific spacer integration. Not only did multiple subdominant strains emerge from this single S. thermophilus colony, but sequences of the original wild-type phage were not detected in cultures within one week of acquired bacterial immunity. The phages that did circumvent early immunization events had SNPs and deletions concentrated within or near their proto-spacer.
In a truly remarkable case of immune evasion strategy, functional CRISPR loci also exist within phage genomes themselves. In 2013, Andrew Camilli’s group described a novel phage-encoded CRISPR/Cas system that targets a bacteriophage inducible chromosomal island-like element (PLE) in the bacterium Vibrio cholerae. During ICP1-phage infection, the PLE circularizes and prevents ICP1 replication. However, ICP1s cultured from cholera patient stool samples had evolved specific, functional immunity against the vibrio by incorporating new PLE-spacers into their CRISPR loci. What is exceptionally fascinating about this example is that outside of the human host, V. cholerae evolves resistance to ICP1 by mutating the O1 antigen. This comes at a significant cost, however, as maintaining virulence requires the O1 antigen. Thus ICPl takes advantage of the context of human infection to prey on V. cholerae.
Collectively, studies in bacteria-phage interactions have shown that both sides achieve very rapid diversification of population immunity in a variety of environmental contexts—the hallmark of an evolutionary arms race.
TO CRISPR OR NOT TO CRISPR
CRISPR/Cas systems are found in ~90% of archaea and ~50% of sequenced bacteria. The system is thought to have originated in the extremophiles, horizontally transferred to related thermophilic bacteria, and subsequently moved to other species of bacteria. The benefits of evolving this adaptive immune system are obvious. However, in this context, it may seem puzzling that “only” 50% of bacteria have CRISPR/Cas systems. Why aren’t CRISPR loci represented as broadly across nearly all bacteria as they are in archaea?
To address this question, one must consider the many lifestyle choices that bacteria have made over the course of evolution. A significant number of bacterial species have adapted to specific host environments, whether symbiotic or pathogenic in nature. These require unique strategies for colonization and tolerance or evasion of the host immune system. The remarkable diversity and metabolic capabilities that allow bacteria to prosper in this type of ecosystem are in part achieved by considerable genomic flexibility; bacteria can expand or contract the size of their genome to manage genetic homeostasis and lifestyle phenotypes. Importantly, they can acquire beneficial genetic material from exogenous plasmids, phages, and transposons (collectively known as mobile genetic elements, MGEs), which often carry antibiotic resistance cassettes, virulence factors, and pathogenicity islands.
Now, here, you see, it takes all the running you can do, to keep in the same place!” – Through the Looking Glass
Consequently, there are evolutionary costs to playing a stacked defense. An overbearing CRISPR system, while providing protection against viral infections, can simultaneously act as a barrier to acquiring beneficial genetic material needed for pathogenic phenotypes. Pathogenic bacteria tend to dampen the use of their CRISPR systems (at least for antiviral or MGE immunity), while others lack the loci completely. Enterococci genomes are made up of 25% MGEs but contain 0% CRISPR loci, whereas several Streptococcus species suffer loss of virulence when their CRISPR defenses are activated. There is also a negative correlation between the number and diversity of CRISPRs and the presence of plasmids and prophages in pathogenic species. Whether pathogens that lack CRISPR loci once had them and actively lost them, or never acquired them and managed to survive regardless remains to be fully understood.
Therefore, it is intriguing to speculate whether ancient bacteria that failed to receive CRISPR/Cas systems may have been better situated to evolve a pathogenic lifestyle with the help of MGEs. However, not all pathogenic bacteria view CRISPR systems as harmful to their existence. Certain species have in fact adapted CRISPRs to play offense, by using crRNAs to regulate virulence genes and evade host defenses. In Francisella novicida, for example, CRISPRs actively down-regulate a lipoprotein that triggers pro-inflammatory responses in the host. There is also emerging evidence that CRISPR loci control the regulation of genes that contribute to colonization, biofilm formation, swarming motility, and pathogenicity. Beyond host-phage co-evolution, CRISPR systems are also intimately linked with community structuring and biogeographic patternings in microbial mats, acidophilic biofilms, and hot spring microbiota. In sum, depending on the niche environment, bacteria employ (or avoid) CRISPR/Cas systems for a variety of biological and evolutionary processes.
EXPLOITING THE ARMS RACE FOR MICROBIOME AND ANTIMICROBIAL RESEARCH
The exploration of bacterial and phage evolutionary mechanisms has far-reaching benefits. Scientists are now engineering approaches that exploit these mechanisms to selectively combat drug-resistant or detrimental gut bacterial species resident within heterogeneous, healthy microbial populations. This is achieved by designing Cas9 nucleases and different CRISPR guide RNAs to target sequences unique to specific bacterial genomic elements. The killer-Cas9 plasmids are delivered to the offending populations by hijacking the natural processes of bacteriophage predation or conjugation (horizontal transfer of plasmids among bacteria).
In a study by Bikard et al in 2014, Cas9 was reprogrammed to target virulence and antibiotic resistance genes in Staphylococcus aureus. The nuclease, delivered by bacteriophage, destroyed S. aureus plasmids harboring antibiotic resistance genes, and effectively immunized avirulent staphylococci against other plasmid-based resistance genes. That same year, another research group (Cirotik et al) demonstrated knockdown of specific bacterial strains within a Galleria mellonella infection model. This result is particularly promising for developing ways to better manipulate microbiomes in other research models.
Indeed, the healthcare and economic potential for customizable antimicrobials is incredible. With further development, these strategies could treat multidrug-resistant infections with unprecedented specificity, could enable specific remodeling of human gut microbiota, and could even be harnessed to remove costly contaminating microbes in dairy and agricultural industries.
THROUGH THE LOOKING GLASS: THE FUTURE AHEAD
The discovery of CRISPR, what was once considered a curious phenomenon in bacterial genomics over two decades ago, has ushered in an explosive era of research. There are several fascinating sides to the CRISPR/Cas story—some that control bacterial ecology, immunity, and evolution, and others that drive diverse phage immune evasion strategies and genome co-evolution. Continued research in these areas will broaden our fundamental understanding of bacterial and viral biology, which in turn will yield additional, yet unforeseen, applications in research and healthcare.
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