Deliver CRISPR editing tools with bacteriophages to precisely edit specific bacteria of the soil microbiota

Scientists at North Carolina State University have developed a way to precisely edit individual genes
of a target bacterial species in a microbial community using bacteriophages that attack bacteria.
The findings were published in the Proceedings of the National Academy of
Sciences.

"We think it's a mechanism
that helps the microbiome.
" We can make changes to one particular bacterium without damaging the rest of the microbiome," said Dr.
Rodolphe Barrangou, a distinguished professor of food, bioprocessing and nutrition at North Carolina State University and editor-in-chief
of CRISPR Journal, GEN's sister publication.
"This is a proof of concept that can be applied to any complex microbial community to promote plant health and gastrointestinal health – microbiomes that are important
for food and health.
"

First, the authors investigated whether bacteriophages T7 and lambda could engineer the CRISPR mechanism to deliver to E.
coli
.
Phages like lambda can integrate their genetic material into the genetic material of the host bacteria and co-replicate
with the host bacteria.
Such a viral life cycle is called "lysogenic"
.
The researchers noted that lambda lysophiles are effective
when expressing exogenous base editing tools in E.
coli to knock out specific genes.
The results showed that both engineered phages — T7 and lambda — were able to deliver payloads to E.
coli hosts that expressed bacterial fluorescent genes and manipulated bacterial resistance to antibiotics
.

The researchers used engineered lambda phages to deliver cytosine base editors (CBEs) into E.
coli in test tubes and microbial communities containing the bacteria of interest, altering a base
in E.
coli DNA with unique sensitivity and precision.
To simulate a microbial community model environment containing a range of bacteria, the research team used EcoFAB filled sterile white quartz sand as synthetic soil, adding three different types of bacteria to investigate whether bacteriophages could specifically localize E.
coli
in the system.
The results showed that engineered lambda phages could efficiently attach to E.
coli and edit their genomes
.

Understanding the interactions of soil rhizospheres is important to ensure the sustainability of food resources – soil rhizospheres are soil layers
affected by root exudates and associated microorganisms.
This study lays a foundation
for the use of soil microbial communities to control the composition and function of plant-associated bacteria.
These findings can help design ecosystems to promote plant growth and health, which is essential
for sustainable agriculture.

The method developed in this study is somewhat "ironic" because, biologically, bacteria use the CRISPR-Cas system to fight back against attacking viruses
.
Biotechnology enables the CRISPR-Cas system to edit genes in animals and humans
.
But in this study, the researchers in turn loaded the virus with the CRISPR-Cas mechanism to alter the behavior
of certain target bacteria present in the microbiome.

"Viruses are very good at delivering payloads
.
Here, we use a bacterial virus, a bacteriophage, to deliver CRISPR to bacteria, which is ironic because bacteria usually use CRISPR to kill viruses
.
"In this case, the virus attacks E.
coli by delivering DNA to it
.
It's
like treating the virus like a syringe.
Dr Matthew Nethery, lead author of the study, said: "We are here using the base editor as a programmable switch
in the E.
coli gene.
Using such a system, we can make highly precise single-base alterations to the genome without the need for CRISPR-Cas to target the double-stranded DNA breaks
typically involved.
”

Dr.
Trent Northen, a scientist at the Department of Energy's Lawrence Berkeley National Laboratory and Barrangou's team, said, "This technology will enable our team and others to discover the genetic basis
of key bacterial interactions with plants and other microbes in highly controlled laboratory settings such as EcoFABs.
" Barrangou's team intends to test phage CRISPR technology
with other soil-associated bacteria in future experiments.