Unlocking the future of gut health: Researchers spotlight CRISPR-Cas systems for probiotic engineering

18 Dec 2023 --- Researchers from Huazhong Agricultural University in China set out to promote the advancement of probiotic research by reviewing the latest advances and hurdles in CRISPR-Cas systems discovered in microorganisms. They propose a design route for CRISPR systems related to genetically engineered probiotics.

While CRISPR-based genome editing has progressed, public concerns about engineered probiotics remain. Microbial biocontainment is one of the most significant challenges. The researchers suggest technologies such as kill switches to construct probiotic chassis with controllable viability.

The high efficiency, flexibility and specificity of these systems have improved genome editing of probiotics in recent years. The scientists summarized the classification and distribution of CRISPR-Cas systems in probiotics, including the editing tools developed as a result of these systems.

“The intake of probiotics has been gradually demonstrated as an effective strategy to prevent or mitigate diseases in humans. Among various probiotics evaluated, genetically modified probiotic strains can have stronger or new properties and exhibit greater research and application value,” says Nan Peng, professor at Huazhong Agricultural University and principal investigator of the study.Scientists propose a design route for CRISPR systems to overcome challenges in the genetic engineering of probiotics.

Defense mechanisms for bacteria
In their natural forms, CRISPR-Cas systems are defense tools for bacteria against viruses or phages. When a bacterium survives a viral attack, it stores some viral DNA in the form of CRISPR sequences.

Various genome editing techniques have been developed and applied in recent years to explore probiotics’ mechanisms and functional modifications. The research team summarized the critical CRISPR-Cas systems used to edit genes in several probiotic organisms, including lactic acid bacteria, yeast and Bacillus. The findings were published in BioDesign Research.

The researchers investigated recent developments in therapeutic applications of probiotics that were genetically modified through the CRISPR-based systems. “As important genome editing tools, CRISPR-Cas systems have opened the window to new improvements in genome editing dedicated to probiotics thanks to their high efficiency, flexibility and specificity,” explains Peng. 

“Undoubtedly, the use of engineered probiotics to promote the development of animal and human health industries will face significant opportunities and challenges in the future.”

Circumventing antibiotic-resistant bacteria
When probiotics — live bacteria and microorganisms that influence the gut microbiota — are consumed in the right amounts, they promote gut health, support the immune system and enhance metabolism.

For example, Escherichia coli (E. coli) can eliminate antibiotic-resistant bacteria in the gut and protect it against harmful E. coli infections. Yeast is used to treat inflammatory bowel disease, and Bacillus subtilis is used to regulate metabolism and help prevent obesity.

A few of these strategies include the development of CRISPR tools to reduce errors during cleaving and gene insertion, optimizing the editing system, using alternative Cas9 proteins (such as dead-Cas9) and ensuring the stability of engineered strains.

If the same virus appears again, the bacterium produces “guide RNA” molecules based on the CRISPR sequences — stored as memories. These molecules guide Cas proteins, which act like molecular scissors, to cleave and neutralize the targeted viral DNA. Scientists have found methods to leverage these molecular mechanisms as toolkits for precise gene editing.

However, it is often challenging to use probiotics in their existing form because of the different effects of various strains on the health of individuals. Finding concrete evidence to support the benefits is also complicated as a result. Genetic engineering, particularly the CRISPR-Cas editing system, has allowed researchers over the past decade to address these challenges.Editing probiotics facilitates the customization of probiotic organisms for precise health benefits.

Modification tools and obstacles
Genome editing technologies, including transcription-activator-like effector nucleases, zinc-finger nucleases, inducible plasmid self-destruction and assisted genome engineering paved the way for the functional modification of probiotics.

Genome engineering and synthetic biology have facilitated the construction of novel probiotic strains with desired functions for treating metabolic disorders, inflammation and pathogen infection. 

Editing, deleting or introducing specific genes with these tools allows scientists to tailor the activities of probiotic organisms to fit particular health benefits. Genome engineering of probiotics to submit, remove or modify phenotypes will improve their tolerance to stress during food production, promote their survival in the gastrointestinal tract or enhance their function. 

According to the researchers, an obstacle to the clinical application of genetically modified probiotics is safety, such as the immunogenicity of functional proteins and the potential for inflammation caused by preexisting antibodies against CRISPR components. Further research is needed to refine the processes and functionalities. 

Meanwhile, North Carolina State University researchers are analyzing ways to improve the human gut bacterium Bifidobacterium to enhance genetic engineering and improve probiotics. According to researchers, Bifidobacterium is important but challenging to define and is utilized in many probiotics that support healthy microbiomes.

By Inga de Jong