The role of CRISPR in microbiome engineering [Podcast]

How CRISPR-Cas9 helps explore the role of human microbiome in diseases

It is now well known that the human body contains trillions of microorganisms, thereby outnumbering human cells. Collectively called the microbiome, these microorganisms form a symbiosis with the body by regulating the extracellular environment and protecting cells against pathogens. So, it is no surprise that disruptions in the microbiome are closely associated with numerous diseases, from diabetes and obesity to cancer.

One way CRISPR contributes to drug discovery is its implementation to alleviate the disruptions in the microbiome. This is achieved by engineering CRISPR-Cas9 systems to cut specific bacterial DNA sequences to eliminate pathogenic bacteria. SNIPR Biome is one of the companies actively working towards this goal. Dr. Jakob Haaber, Vice President and head of CRISPR and Delivery Technologies at SNIPR Biome, describes the application of CRISPR to pathogenic E.coli elimination: “We target them using our CRISPR that we have programmed to kill E.coli. Currently, the stand out of care for treating bacterial infections is with antibiotics. But there is an increased occurrence of antibiotic resistance, rendering antibiotics ineffective. The advantage of CRISPR is that it does not distinguish between antibiotic sensitive- and antibiotic-resistant bacteria''. He also suggests that CRISPR can specifically target pathogenic bacteria and spare the beneficial ones integral to the healthy gut microbiome, as opposed to antibiotics with detrimental effects on healthy bacteria alongside pathogens.

Advantages of CRISPR and recent developments

According to Richard Fox, co-founder, CEO, and CTO of Infinome Biosciences, the main strength of CRISPR comes from its ability to scale up genome editing. “Instead of random mutations on a small set of target genes, we can now precisely edit an entire pathway or genome, introducing hundreds of thousands of changes.”

Large-scale genome editing gives rise to commercially available cell libraries that researchers can integrate into a high-throughput phenotypic screening. These libraries eliminate the need to manually knockout a large number of genes for phenotypic profiling and propose a solution for the acceleration of drug discovery studies.

Another advantage of CRISPR technologies is the improved specificity. Microbiome gene editing is one of the fields reaping the benefits of specificity. CRISPR systems can be programmed to cut specific bacterial DNA or even remove genetic circuits within bacterial cell walls without killing the cell. Taken together, these advancements drive novel gene therapies for gut microbiota deviations.

In the meantime, CRISPR systems have enabled the manufacturing of smaller genetic constructs for easier packaging into delivery vehicles. Thus, the delivery vehicle can carry multiple components in addition to CRISPR, making up a multifunctional gene-based therapeutic that accomplishes a lot more than just cutting or inserting DNA.

Acceleration in the manufacturing of CRISPR technologies

The gap between synthetic biology and bioengineering is often caused by issues regarding time and cost. Bringing a biomanufacturing solution to market requires large entities with well-built infrastructure, robust automation, lots of people, instrumentation, informatics, and a lot of capital. So, it is not surprising that the development of biologics can take 5-10 years and many millions of dollars. This is quite a risky investment, so pharmaceutical companies often hesitate to partake in it, leaving territories with huge therapeutic potential unexplored.

The setup of the CRISPR-enabled genome editing system is one of the bottlenecks in workflows. The design of large-scale genome engineering systems is quite laborious because one may need to produce minute amounts of donor sequence to target and precisely edit 10,000 loci into a whole genome.

One of the co-founders of Infinome Biosciences, Andrew Garst, came up with a key innovation by pairing the guide sequence that directs the cut with the donor sequence that mediates the repair, thereby reducing wide-genome editing to just a few clicks. Richard Fox believes “such automated design and build systems can create edited cell libraries in less than a week, which is the fraction of the effort it used to take”.

Steps of microbial gene editing

The first step in gene editing for the gut microbiome is the in vitro validation of the CRISPR system. The specificity of the CRISPR editing is tested against a set of bacterial panels representative of the gut microbiome. The aim is to ensure that the CRISPR system targets a predetermined subspecies of bacteria and that the beneficial bacteria are exempted from CRISPR-induced gene editing

Then, the validation carries on to the preclinical and clinical studies. Using genome sequencing techniques, researchers demonstrate that gene editing does not perturb the microbiome in a harmful way. Of course, off-target effects could inevitably occur, as revealed by whole-genome sequencing. The course of action is to monitor the rate of occurrence and ensure that these effects are trivial and do not interfere with gene editing.

Sequencing and phenotypic profiling reveal the set of edits that a genome carries as well as the prevailing strains in a bacterial population. To further validate the success of targeted genome editing, one can subject the bacterial population to environmental stressors and monitor their behavior. This ensures that the gene editing confers the bacteria to the desired properties, for instance the ability to grow under an environmental stress such as hypoxia.

With accelerated CRISPR workflows, researchers can elicit the set of beneficial gene edits amongst hundreds of thousands of gene knockouts. 

CRISPR systems also unlock the flexibility to enrich or deplete the same bacterial strain depending on the specific research need. For example, researchers at Infinome implemented CRISPR to engineer E.coli strains to scale up lysine production, a critical amino acid used as a food additive. On the other hand, SNIPR used the same system to eradicate detrimental E.coli strains from the gut of hematological cancer patients to prevent bloodstream infections.

Looking ahead to the future of CRISPR

As mentioned before, CRISPR empowers the fight against pathogenic bacteria. It has been proven several times that conventional antibiotic treatments disrupt the gut microbiota by targeting both harmful and beneficial bacteria. CRISPR can help enhance the specificity of bacteria to maintain the balance of the human gut microbiota.

Another exciting prospect driven by CRISPR is the ability to engineer bacterial strains with therapeutic potential. This can be used to add genetic functions to the microbiome through engineered bacteria, which can express a specific enzyme or metabolite that the body previously lacked.

Lastly, the successful implementation of CRISPR in biomedical and bio-industrial engineering relies on the ability to make combinatorial edits, also known as “DNA shuffling”. Especially in large microbial systems, the ability to introduce multiple edits to reduce the number of screening rounds will be paramount to implement bio-industrial production. 

Molecular Devices helps scientists by providing cutting-edge technologies that accelerate the immediate goal of CRISPR-Cas9 genome editing. An accurate library screening and selection of the CRISPR-edited hits is key within microbiome engineering workflows, allowing for faster availability of the biomanufacturing product to the market.