Harnessing the potential of CRISPR-Cas9 for Gut Microbiome Gene Editing

The microbiota colonizing the gut or skin is indeed very complex. To understand the molecular underpinnings of the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of tools to precisely perturb this complex community.

After years’ of monitoring the human microbiota, researchers are now leveraging the potential of CRISPR-Cas9, a DNA-editing system, to turn the tables on gut microbiome gene editing.

In the landmark study published in Cell, the researchers from University of California, San Francisco have successfully used the CRISPR-Cas9 for manipulating the genome of bacteria, E.coli, colonizing the mammalian gut.

This work provides a valuable step toward establishing a toolkit for microbiome editing that could eventually pave the way for better prognosis and treatment of various gut-related diseases.

Given the tremendous diversity within both the bacterial and viral components of the human gut microbiota, the researchers have used a tripartite model system that builds upon tools for the genetic manipulation of a bacteriophage, and its bacterial target coupled to an experimentally tractable mammalian host.

Related Article: Scientists Identify Antidotes To Overcome Collateral Damage of Antibiotics on Gut Microbiota

Engineering a Virus to Wreck E. coli

In a first, the researchers have successfully used the bacteriophage M13, a single-stranded DNA (ssDNA) filamentous virus for delivery of DNA to E. coli cells colonizing the mouse gut.

M13 naturally attacks E. coli but typically does not survive well inside the gastro-intestinal tract. To add to their success, the researchers spliced an antibiotic resistance gene into the DNA that M13 would deliver to the E. coli cells, allowing the M13 virus – and the CRISPR-Cas9 system it carried – to spread more easily in the mouse gut.

The authors also showed that the engineered M13 carrying CRSIPR-Cas9 enabled the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization in the gut.

Although full eradication of the targeted strain was difficult to achieve due to the ability of E. coli cells to survive Cas9-induced double-stranded breaks repair by homologous recombination, the results were dramatic. 

After gene editing, the targeted strain quickly began to disappear. In about two weeks, it represented only one percent of the monitored cell population.

This paves the way for advancements in precision gene editing of the gut microbiome by specifically targeting harmful strains of E. coli while leaving ‘helpful ones’ undisturbed.

The researchers opined that the growth in competitive colonization increased the efficiency of targeting a strain for depletion due to the resulting growth impairments in the targeted strain.

In addition, the investigators also demonstrated that the M13-delivered CRSIPR-Cas9 induces chromosomal deletions encompassing the targeted gene in E. coli colonizing the mouse gut. An advantage of this approach is that the deletion of a single genomic locus is unlikely to have as large an impact on the rest of the gut microbiota than if the strain were to be removed entirely.

A Stubborn Strain

Interestingly, the investigators also detected a wide range of chromosomal deletion sizes in E. coli cells, highlighting the ability of bacteria to survive large deletions and opening up the potential for the in vivo removal of entire biosynthetic gene clusters or pathogenicity islands. 

However, multiple mechanisms enable E. coli to escape targeting, including loss of the spacer in the CRSIPR array, target site mutations, or even deletion of the entire CRISPR-Cas9 system.

Nevertheless, the researchers envision that it may be feasible to deliver more complex genetic circuits to E. coli, with the goal of boosting metabolic pathways beneficial to its mammalian host or altering immune function.

However, the application of these approaches will require a renewed effort to isolate and characterize bacteriophages that target strains of interest.

Pioneering New and Improved Microbiome-Altering Tools

Despite certain limitations in the study such as low rates of gene delivery, lower absorption rates of M13 phage, use of antibiotics for selection, including others, this breakthrough discovery to alter the DNA of the microbes already in the gut facilitates the study of the microbiome in a more controlled way. It provides a lead to the discovery of similar tools for a more diverse panel of bacteria found within the human microbiota. Also, this pioneering study in the guts of mammals paves the way for precise research and treatment of a broad range of microbiome-related ailments. 

Author

Isha Kapoor