The Phage


Category: CRISPR-Cas

Kimchi LAB Strains that defeat phage infection

When you talk about fermented foods, kimchi stands out not only as a culinary delight but also as a treasure trove of microbial wonders. One might typically view viral resistance in bacteria as a cause for concern, as it may halt therapy. In the context of kimchi, this resistance shines brightly, enhancing the culinary experience with its remarkable flavors. These bacteria play a crucial role in fermenting kimchi, making them essential for its production. If these bacteria were to be attacked by bacteriophages, the entire process of kimchi production could come to a standstill or reduce production, depriving us of the delightful flavors that make kimchi a culinary delight.

Produced through low-temperature fermentation without pre-sterilization, kimchi hosts a diverse microbial community, shaping its unique flavors and textures. Researchers at the World Institute of Kimchi have delved into this microbial universe, uncovering fascinating insights into the dominant lactic acid bacteria (LAB) species that safeguard kimchi against viral invasions.

In a pioneering study published in the journal Food Microbiology, scientists explored long-term fermented kimchi stored at low temperatures, collecting samples from various regions in South Korea. Their meticulous analysis revealed a specific LAB strain, Pediococcus inopinatus, as the stalwart defender in over 88% of the studied kimchi samples. What sets P. inopinatus apart is its well-developed clustered regularly interspaced short palindromic repeats (CRISPR) system, a sophisticated prokaryotic adaptive immune system that combats phages, and viruses that infect and replicate within bacteria.

Through whole-genome sequencing analysis, the researchers unearthed the unique genetic traits of P. inopinatus. This exceptional LAB strain boasts an abundance of the csa3 gene, responsible for the transcription factors that activate cas genes. Consequently, P. inopinatus stores a wealth of genetic information about phages, enabling it to fend off viral invasions effectively. After the initial phage infection, P. inopinatus becomes adept at preventing subsequent infections, ensuring the stability of the kimchi fermentation process.

This research not only sheds light on the intricate microbial dynamics within kimchi but also paves the way for future studies exploring the broader implications of these findings. As the world continues to grapple with viral challenges, the humble kimchi emerges as a source of inspiration, offering valuable lessons in microbial resilience and antiviral defense mechanisms. The microbial guardians of kimchi, particularly P. inopinatus, stand as a testament to nature’s ingenuity, reminding us of the boundless wonders that the microbial world has to offer.

To read more please visit Mun, S. Y., Lee, W., Lee, S. Y., Chang, J. Y., & Chang, H. C. (2024). Pediococcus inopinatus with a well-developed CRISPR-Cas system dominates in long-term fermented kimchi, Mukeunji. Food Microbiology, 117, 104385.

Cell free synthesis of bacteriophages proved more efficient

Many people are now familiar with phage therapy, a fascinating approach that harnesses viruses to combat drug-resistant bacteria. While this therapy boasts numerous benefits, there are some significant drawbacks that hinder its widespread adoption.

In contrast to antibiotics, which can be easily mass-produced in standardized production lines, phage therapy is often personalized for each case. This individualized approach poses challenges, as it can be time-consuming to prepare bacteriophages compared to the ready availability of antibiotics. To address this issue and enhance efficiency, scientists from Tulane University have successfully synthesized bacteriophages independently from bacterial cell cultures. This breakthrough offers a promising glimpse into a future where standardization can increase the efficiency in delivery of phage therapy.

In vivo bacteriophage replication and cell free bacteriophage synthesis (CFBS)
The performance of CFBS yields can be significantly affected by the gene expression background within the TXTL source. When bacteriophage T7 infects E. coli BL21, the number of offspring it produces is greatly influenced by the genetic makeup of the host at the time of infection. The knockdown (KD) or overexpression (OX) of specific genes can result in a variety of effects on T7 progeny yield in comparison to wild-type BL21 (WT), which can be positive, negative, or neutral (a). Furthermore, by altering the genetic context of the transcription and translation (TXTL) machinery derived from cell lysates, it is possible to modulate the synthesis of cell-free bacteriophages (CFBS) (b).

Cell-free bacteriophage synthesis: How was it done?

In a technique known as Cell-free Bacteriophage Synthesis (CFBS), scientists generated phage virions in a controlled environment outside of traditional laboratory settings that involve live bacteria. This innovative process replicates the natural in vivo phage production by utilizing cellular components extracted from bacterial lysates, such as transcription/translation machinery (TXTL), which is then combined with specific reagents to produce proteins based on the genetic instructions found in the DNA of the phage.

To put it simply, CFBS allows us to create phage virions without using live bacteria in a lab. It’s like mimicking what happens inside a living cell but in a test tube, using cellular components and specific substances to make the proteins needed for the bacteriophage’s replication when it infects a bacterial cell.

CELL free bacteriophage synthesis (CFBS) flow chart

In this research, a process called TXTL (Transcription and Translation) was carried out using genetically modified E. coli BL21 to improve the production of the T7 bacteriophage in vitro through CFBS. To do this, they made alterations to the genetic makeup of these E. coli cells by manipulating 18 specific bacterial genes.

To manipulate these genes, the researchers employed a technique known as inducible CRISPR (read more about CRISPR Cas and other defense systems here) interference (CRISPRi), which involves an enzyme called Cas12a . This Cas12a enzyme, taken from a different bacterium (F. novicida), lacks nuclease activity, and it was used to identify the specific genes involved in T7 phage replication as either having a positive or negative impact.

Once the relevant genes were identified, the scientists further modified the TXTL’s genetic background for CFBS. They did this by either increasing the activity of genes that had a positive effect (overexpressing) or reducing the activity of genes with a negative effect (repressing). This process allowed them to fine-tune the genetic environment within the TXTL to improve the production of the T7 bacteriophage in vitro.

Wonderful results

The results of this study were quite promising. They found that by making specific genetic modifications during the process of producing the T7 bacteriophage using CFBS, they were able to significantly increase the yield of these phages in vitro, sometimes by as much as 10 times.

The improvements were achieved through the following changes:

  1. Overexpression of the translation initiation factor IF-3 (infC).
  2. Enhanced levels of small RNAs OxyS and CyaR.
  3. Repression of the RecC subunit exonuclease RecBCD.

Translation initiation factors like IF-3 are proteins that play a crucial role in initiating the translation of genetic information into proteins, which is a fundamental step in building proteins. By manipulating these factors and small RNAs, the researchers optimized the process of creating T7 phages in the lab.

This is significant because it means that we can produce T7 bacteriophages more efficiently, potentially up to 10 times as effectively, by carefully adjusting the genetic components involved in their creation. By doing so, we can address some of the bottlenecks in phage manufacturing, making it easier and more practical to produce these viruses. This, in turn, can help lower barriers to the widespread use of phage therapy, which holds great promise as an alternative to traditional antibiotics in combating drug-resistant bacteria.

Reference and credits

Every image featured in this article originates from the same study that provided the information used in this article. To delve deeper into the details, please refer to the published study cited below. Brooks, R., Morici, L., & Sandoval, N. (2023). Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield. ACS Synthetic Biology12(8), 2418-2431.

Phage-Powered Evolution Unleashes Compact and Mighty Prime Editors

In the mesmerizing world of genetic engineering, prime editing has emerged as a shining star, promising to rewrite the very fabric of life with pinpoint precision. But here’s the plot twist: a team of relentless scientists at the Broad Institute of MIT and Harvard have summoned the powers of phages and protein engineering to birth a new generation of prime editors that are not just efficient but astonishingly compact.

The Genesis of Prime Editing

Prime editing, akin to the artistry of a genetic sculptor, was unveiled in 2019 by the visionary David Liu and his team. This editing technique entails genome modification through search-and-replace mechanisms, all achieved without the need for double-strand breaks or donor DNA.  This molecular wizardry combines a tweaked Cas9 protein, an engineered prime editing guide RNA (pegRNA), and a modified reverse transcriptase enzyme. Together, they form a potent triumvirate capable of performing DNA edits with laser-like precision.

Epic Evolution: Phages to the Rescue

Enter the heroes of our tale: bacteriophages, the viruses that prey on bacteria. The scientists harnessed the evolutionary prowess of these tiny warriors through a technique called phage-assisted continuous evolution (PACE), first forged by David Liu in 2011. Picture this: a battlefield in the lab where only the bacteriophages that bear the most desirable genetic traits can survive and thrive, replicating faster than they can be wiped out.

In this grand showdown, the bacteriophages carrying the secrets of prime editing faced countless generations of evolution. The result? Brand new, turbocharged reverse transcriptases that turbocharge prime editing to unparalleled heights.

Prime Editors: Not All Heroes Wear Capes

Here’s the twist in our tale: Not all prime editors are cut from the same genetic cloth. The evolved prime editors have their own unique superpowers. Some excel in adding or swapping just a few DNA letters, while others stumble in these tasks but soar when it comes to inserting long DNA sequences. This revelation shattered the notion that one prime editor could rule them all.

Expanding the Editing Universe

With their newfound superpowers, the prime editors, now known as PE6a through PE6g, have blown the lid off previous limitations. These titans are two to 20 times more efficient than their predecessors, raising the banner of hope for therapeutic applications higher than ever before.

Their versatility shines bright as they effortlessly insert lengthy DNA sequences—ranging from 38 to 108 base pairs—into the genomes of human cells. Even the once-untamable frontier of live mouse brains has succumbed to their precision, with editing efficiency soaring to astonishing heights.

Structure (predicted by AlphaFold) of a new prime editor developed by the David Liu lab.
The David Liu lab’s new prime editor’s structure, as predicted by AlphaFold. Credit: Harvard University and MIT’s Broad Institute

Prime Editors: Travel Light, Edit Right

But there’s more to this story. These newfound prime editors have slimmed down, shedding excess genetic baggage. This means they can squeeze into delivery systems that previously dared not accommodate them. This improved agility opens the door for prospective therapies in animal models as a necessary preliminary step before human studies.

You can find the complete article by following this link, and for additional updates on phage-related technologies and other related news, please explore our news section (The Phage News section).

Article reference: Jordan L. Doman et al, Phage-assisted evolution and protein engineering yield compact, efficient prime editors, Cell (2023). DOI: 10.1016/j.cell.2023.07.039

CRISPR-based Phage Therapy Shines in the First Human Trial

SNIPR Biome, a Danish microbiome technology company, has unveiled promising results from the initial phase of their groundbreaking CRISPR-based phage therapy targeting Escherichia coli in the gastrointestinal tract.

Imagine a team of microscopic superheroes armed with advanced DNA editing technology. That’s SNIPR001, a powerful blend of four bacteriophages (tiny viruses that specialize in obliterating bacteria) equipped with CRISPR/Cas technology. Its mission? To selectively eradicate E. coli in the gut, even the stubborn antibiotic-resistant strains. And guess what? SNIPR001 is already showing remarkable potential.

After acing its animal trials with flying colors, the remarkable product, SNIPR001, has emerged as a potential game-changer. The exciting breakthrough in animal trials laid the foundation for its momentous journey, leading to the eagerly awaited human trial. Now, with the spotlight firmly fixed on this innovative marvel, we delve into its fascinating story, tracing its path from animal triumphs to the groundbreaking human trial.

The eagerly anticipated phase 1 trial, conducted on 36 individuals, aimed to assess the safety and effectiveness of SNIPR001. The results were nothing short of impressive. Participants who received oral doses of SNIPR001 over a span of 7 days experienced only mild to moderate side effects, demonstrating excellent tolerability. But that’s not all. The treatment also exhibited a numerical reduction in gut E. coli levels, highlighting its ability to combat this problematic bacterium.

This groundbreaking therapy is set to provide a lifeline to patients grappling with hematologic cancers, such as lymphoma and leukemia. These individuals often undergo hematopoietic stem-cell transplants, leaving them vulnerable to bloodstream infections resulting from E. coli moving from the gut into the blood. Unfortunately, the most common antibiotic treatment, fluoroquinolones, is rendered ineffective against fluoroquinolone-resistant E. coli strains. Moreover, it tends to harm the gut microbiome, wiping out the beneficial bacteria crucial for overall health.

“With the combined killing effects of bacteriophages and CRISPR-Cas technology, SNIPR001 has demonstrated the ability to target and eliminate antibiotic-resistant E. coli strains in the gut, providing a safe alternative to traditional treatments that do not work against antibiotic-resistant strains, while sparing the rest of the gut microbiome.”

Christian Grondahl, co-founder, and CEO of SNIPR Biome

Buoyed by these encouraging results, SNIPR Biome is eagerly marching forward to conduct further clinical studies. Their ultimate goal? To improve patient outcomes by revolutionizing the field of antimicrobial resistance. The next phase of trials will focus on investigating whether SNIPR001 can effectively reduce the incidence of E. coli bloodstream infections in cancer patients.

In recognition of its groundbreaking potential, SNIPR Biome secured $3.9 million in funding from CARB-X (the Combating Antibiotic Resistant Bacteria Biopharmaceutical Accelerator) in May 2021. This financial support serves as a testament to the growing excitement surrounding SNIPR001 and its potential to reshape the landscape of microbial therapies.

As SNIPR Biome embarks on the next chapter of its groundbreaking journey, there is a glimmer of hope that this innovative therapy will provide a safe and effective solution against antibiotic-resistant strains of E. coli, all while preserving the delicate balance of the gut microbiome.

Revolutionizing Infection Treatment: SNIPR Biome’s CRISPR-armed Phage Therapy Targets Antibiotic-Resistant E. Coli in Gut

SNIPR Biome ApS has made a groundbreaking discovery in the field of microbiology, paving the way for a new era of microbial gene therapy. Nature Biotechnology has recently published the results of SNIPR’s preclinical work on SNIPR001, a revolutionary CRISPR-armed phage therapeutic designed to specifically target and remove E. coli, including antibiotic-resistant strains, in the human gastrointestinal tract.

The increasing prevalence of antibiotic-resistant pathogens poses a major global challenge to the medical community. Current medical practices rely on increasingly aggressive therapies, which can sometimes result in patients experiencing life-threatening infections caused by antibiotic-resistant bacteria. This issue is often underestimated in contemporary healthcare systems, and the threat of antimicrobial resistance could lead to significant healthcare challenges in the future.

SNIPR001 is a potential game-changer, designed to prevent infections from spreading into the bloodstream by specifically targeting and eradicating E. coli in the gut. This breakthrough therapy has the potential to revolutionize the way we prevent and treat infections and could serve as a model for developing similar therapies that target other life-threatening pathogens. SNIPR Biome’s research has been validated through the publication of their findings in Nature Biotechnology, opening up exciting possibilities for the future of microbial gene therapy.

An overview of the SNIPR001 creation process acquired from the original paper

Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli, SNIPR Biome screened a library of 162 wild-type (WT) phages and identified eight phages with broad coverage of E. coli, complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. The selected phages were then engineered with tail fibers and CRISPR-Cas machinery to specifically target E. coli. SNIPR Biome’s research shows that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli, and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is currently undergoing a Phase 1 trial in the US to evaluate its safety and efficacy in reducing E. coli in the gut without disturbing the overall gut microbiome (NCT05277350).

Jumbo phage chimallin (ChmA) protein: The answer to CRISPR-Cas bacterial defense system?

Chimallin covering phage genome
Loughlin et, 2020

Jumbo phages are bacteriophages (phages) with genetic information (genomes) larger than 200 kilobases. These phages are known as “jumbo” because they have a huge genome size. In addition, they exhibit several novel characteristics not seen in smaller genome phages, which distinguish jumbo phages in aspects of genome and virion structure, progeny propagation, and evolution. According to some studies, jumbo phages can be useful in various ways in this era of antibiotic resistance. They have proven to be more effective than other phages, particularly when dealing with bacterial defense systems and infectivity.

Bacteria encode a plethora of defenses against foreign nucleic acids, and mechanisms like restriction-modification and CRISPR-Cas systems that target invading bacteriophage genome sequences are also present. In rebuttal, some families of jumbo bacteriophages protect their replicating genomic information in a nucleus-like compartment while excluding host defense factors. However, the main structure and composition of this compartment are unknown. According to the University of California, San Diego researchers, a jumbo phage nuclear shell primarily comprises one protein, Chimallin (ChmA). This protein self-assembles as a flexible sheet into closed micrometer-scale compartments growing a shield around the genetic material.
The genomic content of these Jumbo phages is diverse enough to obstruct the detailed comparative analysis that can benefit the study, use, and manipulation of smaller phages of which their genomes are abundantly available in databases. Despite this, there is still a relationship between Jumbo and smaller phages in their genomic and physical structure, which provides a link between the two. Understanding the uniqueness that will be exploited by transferring the better-known genome organization’s knowledge and evolutionary mechanisms which are seen in smaller phages to jumbo phages is critical. The Chimallin protein and many other characteristics can be implemented directly using jumbo phages for therapy and other applications or indirectly through recombination technology by enabling other smaller genome phages to have a “special-hybrid” ability. 
  • Yuan Y, Gao M. Jumbo Bacteriophages: An Overview. Front Microbiol. 2017 Mar 14;8:403. DOI: 10.3389/fmicb.2017.00403. PMID: 28352259; PMCID: PMC5348500.
  • Laughlin, T.G., Deep, A., Prichard, A.M., et al. Architecture and self-assembly of the jumbo bacteriophage nuclear shell. Nature 608, 429–435 (2022).