Author: İrem Eroğlu, Editor: Ece Güven, Redactor: Ayşe Naz Cerit
Observing the effects of genetic mutations on the phenotype is the first way to understand gene function in microorganisms. This classical path requires manipulation of the living in isolation, which is a long road. There are three limitations to this approach. Firstly, most of the species have not been isolated in the laboratory, which makes use of conventional genetic tools unlikely. Secondly, most of the genes that play a role in interactions between microorganisms have not yet been found. Lastly, when microorganisms are isolated and grown in the laboratory, they can show adaptation so fast that they hide their ‘wild-type’ phenotype.
In the study of Rubin et al., individuals in microbial communities were targeted to be manipulated for site-specific genome editing without requiring pre-isolation or engineering. Two applications were developed by researchers, environmental transformation sequencing (ET-seq) to identify organisms that can be traced in a microbial community and DNA-editing all-in-one RNA-guided CRISPR–Cas transposase (DART) systems to insert DNA into the targeted genomic locations of the identified organisms. In this way, they achieved species- and locus-specific gene editing in the gut microbiota and a synthetic soil community.
To evaluate the accessibility of each species for acquiring an exogenous DNA by ET-seq, a mariner transposon (a randomly integrating mobile genetic element) was administered. After DNA extraction from the community, they quantified and located insertions in each species. Then, they applied metagenomic sequencing to measure the abundance of each organism in the community and find relative insertion efficiencies. Five species were determined as genetically accessible in a synthetic soil community. ET-seq was able to successfully identify and quantify genetic manipulations.
For achieving a system that is able to edit targeted genomic regions of an organism in a microbial community, the researchers compared transposition properties of RNA-guided CRISPR-Cas Tn7 transposases from Vibrio cholerae (VcDART) and Scytonema hofmanni (ShDART) by conjugating them into Escherichia coli. After finding that VcDART provides high specificity with low off-target editing and comparable efficiency in transposition, researchers decided to move on with VcDART for the rest of the experiments.
Then, they tested if VcDART used together with ET-seq could allow for tracking fitness of genetic mutants in a synthetic soil community. For that, pyrF gene of Klebsiella michiganensis, which is an endogenous marker, was disrupted by VcDART system and ET-seq was used to quantify relative fitness by comparing growth rates in the presence of growth-inhibitory 5-fluoroorotic acid (5-FOA). The expected results meant that this method is able to assess gene function and fitness in the microbial community. In conclusion, this study shows that ET-seq and DART can be used for species- and locus-specific genome editing by using all-in-one vectors encoding two CRISPR-Cas transposon systems which occur naturally. When using traditional methods, despite all the effort and time to apply complex steps to edit the genome of environmental microorganisms, the study may still fail. Therefore, ET-seq and VcDART are important in accelerating these studies and obtaining more accurate results by eliminating the need for isolation.
Reference: Rubin, B.E., Diamond, S., Cress, B.F. et al. Species- and site-specific genome editing in complex bacterial communities. Nat Microbiol 7, 34–47 (2022). https://doi.org/10.1038/s41564-021-01014-7
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