CRISPR systems have been developed, to date, to edit the genes of one type of cell at a time. Now, a new technique has been characterized that can add or modify genes within a community of many different species simultaneously, opening the door to the idea of “community editing.” The system could be used to edit and track—using a barcode—edited microbes within a natural community, such as in the gut or on the roots of a plant where hundreds or thousands of different microbes congregate.
This work is published in Nature Microbiology, in the paper, “Species- and site-specific genome editing in complex bacterial communities.”
“Breaking and changing DNA within isolated microorganisms has been essential to understanding what that DNA does,” said Benjamin Rubin, PhD, a postdoctoral fellow in the Doudna lab at the University of California, Berkeley. “This work helps bring that fundamental approach to microbial communities, which are much more representative of how these microbes live and function in nature.”
The authors write that the vast majority of bacteria and archaea remain uncultured, “precluding the application of traditional genetic methods to these organisms and their interactions.” In this work, they characterize and validate a strategy for editing the genomes of specific organisms in microbial communities.
The team first developed an approach to determine which microbes in a community are susceptible to gene editing. The screening technique, called ET-seq (environmental transformation sequencing), uses a transposon as a probe that easily inserts randomly into many microbial genomes.
By sequencing the community DNA before and after introducing the transposon, they were able to pinpoint which species of microbes was able to incorporate the transposon gene. In one experiment involving a community of nine different microbes, they successfully inserted the same transposon into five of them using different transformation methods.
They then developed a targeted delivery system called DNA-editing All-in-one RNA-guided CRISPR Cas Transposase (DART) that uses a CRISPR-Cas enzyme similar to CRISPR-Cas9 to home in on a specific DNA sequence and insert a bar-coded transposon. To test the DART technique with a more realistic microbial community, the researchers took a stool sample from an infant and cultured it to create a stable community composed mostly of 14 different types of microorganisms. They were able to edit individual E. coli strains within that community, targeting genes that have been associated with disease.
The researchers hope to employ the technique to understand artificial, simple communities, such as a plant and its associated microbiome, in a closed box. They can then manipulate community genes within this closed system and track the effect on their bar-coded microbes.
While the ability to “shotgun” edit many types of cells or microbes at once could be useful in current industry-scale systems—bioreactors for culturing cells in bulk, for example—the more immediate application may be as a tool in understanding the structure of complex communities of bacteria, archaea, and fungi, and gene flow within these diverse populations.
“Eventually, we may be able to eliminate genes that cause sickness in your gut bacteria or make plants more efficient by engineering their microbial partners,” said Brady Cress, PhD, a postdoctoral fellow in the Doudna lab. “But likely, before we do that, this approach will give us a better understanding of how microbes function within a community.”