Author: University of California, Berkeley December 11, 2021

Two new methods allow simultaneous CRISPR editing of genes in multiple cell types.

So far, CRISPR enzymes have been used to edit the genomes of one type of cell at a time: for example, they cut, delete, or add genes to specific types of cells in tissues or organs, or add to a growing type of cell. Microbes. In a test tube.

Now, the team at the University of California, Berkeley, who invented the CRISPR-Cas9 genome editing technology nearly 10 years ago, has found a way to add or modify genes in communities of many different species at the same time, for the so-called "open door" community editing. "

Although this technology is still only used in laboratory environments, it can be used to edit and track edited microorganisms in natural communities, such as the intestines or plant roots, where hundreds of different microorganisms gather together. When scientists talk about genetic modification of microbial populations, this tracking becomes necessary: ​​for example, inserting genes into gut microbes to solve digestive problems, or changing the microbial environment of crops to make them more resistant to pests.

If there is no way to track gene insertions—in this case using barcodes—the inserted genes may appear anywhere, because microorganisms usually share genes between them.

In order to successfully edit genes among multiple members of a microbial community, scientists at the University of California at Berkeley must develop two new methods: Environmental Transformation Sequencing (ET-Seq), which enables them to assess the editability of specific microorganisms; and DNA editing The integrated RNA-guided CRISPR-Cas transposase (DART), which allows highly specific target DNA to be inserted into a certain position in the genome defined by the guide RNA. The DART system is barcoded and compatible with ET-Seq, so when used together, scientists can insert, track, and evaluate insertion efficiency and specificity. Image source: Jill Banfield Lab, University of California, Berkeley

Benjamin Rubin, a postdoctoral researcher at the University of California, Berkeley, said: “Destroying and altering the DNA in isolated microorganisms is essential for understanding the role of DNA.” “This work helps to introduce this basic method to microbial communities, and these methods are more representative. How these microorganisms live and operate in nature."

Although the ability to "shotgun" multiple types of cells or microorganisms at a time may be useful in current industrial-scale systems—for example, a bioreactor for batch cultivation of cells, a more direct application may be as an understanding of cell structure Tool of. Complex communities of bacteria, archaea, and fungi, and the flow of genes in these different populations.

"Ultimately, we may be able to eliminate the genes that cause intestinal bacterial diseases, or improve the efficiency of plants by modifying their microbial partners," said postdoctoral researcher Brady Cress. "But it is likely that before we do this, this approach will allow us to better understand the function of microorganisms in the community."

Rubin and Cress—both in the laboratory of CRISPR-Cas9 inventor Jennifer Doudna—and Spencer Diamond, a project scientist at the Innovative Genomics Institute (IGI), are the co-first authors of a paper describing the technology emerging today ( December 6)) in the journal Nature Microbiology.

Diamond works in the laboratory of Jill Banfield, a terrestrial microbiologist, a pioneer in the field of community sequencing or metagenomics: shotgun sequencing of all DNA in complex microbial communities, and assembling the DNA into the complete genome of all these organisms , Some of them may have never been seen before, and many of them are impossible to grow in a laboratory petri dish.

In the past 15 years, metagenomic sequencing has made tremendous progress. In 2019, Diamond assembled 10,000 individual genomes of nearly 800 microbial species from soil samples collected from grassland meadows in northern California.

But he compared this to the census: it provides unparalleled information about which microbes are present in what proportions and what functions these microbes can perform in the community. It allows you to infer the complex interactions between organisms and how they work together to achieve important ecosystem benefits, such as nitrogen fixation. But these observations are only hypotheses; Diamond said that new methods are needed to actually test these functions and interactions at the community level.

"There is an idea of ​​metabolic handoff-no microorganism is performing a large number of metabolic functions, but in most cases, each individual organism is doing a step of a process, and there must be some metabolism between the organisms. Things," he said. "This is a hypothesis, but how do we actually prove this? How do we get to the point where we are no longer just observing birds, we can actually perform some operations and see what happens? This is the origin of community editing."

The research team is composed of Benfield, Professor of Earth and Planetary Sciences and Environmental Science, Policy and Management at the University of California, Berkeley, and Professor of Molecular and Cell Biology and Chemistry at the University of California, Berkeley, Howard Hughes Medical Institute Researcher and co-award Jennifer Dudd Leader Na won the 2020 Nobel Prize in Chemistry for his invention of CRISPR-Cas9 genome editing.

The team first developed a method to determine which microorganisms in the community are actually susceptible to gene editing. The screening technology developed by Rubin and Diamond is called ET-seq (Environmental Transformation Sequencing), which uses transposons or jumping genes as probes to easily insert many microbial genomes randomly. By sequencing the community DNA before and after the introduction of the transposon, they can determine which microbial species can incorporate the transposon gene. The method is based on a technique developed by Adam Deutschbauer, a co-author of Lawrence Berkeley National Laboratory. In an experiment involving nine different microbial communities, they used different transformation methods to successfully insert the same transposon into the five microbes.

Subsequently, Cress developed a targeted delivery system called DNA-editing integrated RNA-guided CRISPR Cas transposase (DART), which uses a CRISPR-Cas enzyme similar to CRISPR-Cas9 to locate specific DNA sequences and Insert a bar chart. Encoding transposon.

In order to test the DART technology with a more realistic microbial community, the researchers collected stool samples from babies and cultured them to create a stable community composed mainly of 14 different types of microorganisms. They can edit individual E. coli strains in the community to target disease-related genes.

Researchers hope to use this technology to understand artificial, simple communities, such as plants and their associated microbiomes, in a closed box. Then, they can manipulate the community genes in this closed system and track the impact of their barcoded microorganisms. These experiments are part of a 10-year project funded by the Department of Energy called m-CAFE. The project is used for soil microbial community analysis and functional assessment, aiming to understand the response of simple grass microbiota to external changes . Banfield, Doudna and Deutschbauer are part of the m-CAFEs project.

Reference: "Species and site-specific genome editing in complex bacterial communities" Authors: Benjamin E. Rubin, Spencer Diamond, Brady F. Cress, Alexander Crits-Christoph, Yue Clare Lou, Adair L. Borges, Haridha Shivram, Christine He, Michael Xu, Zeyi Zhou, Sara J. Smith, Rachel Rovinsky, Dylan CJ Smock, Kimberly Tang, Trenton K. Owens, Netravathi Krishnappa, Rohan Sachdeva, Rodolphe Barrangou, Adam M. Deutschbauer, Jillian F. Banfield, and Jennifer A. Doudna, December 6, 2021, Nature Microbiology. DOI: 10.1038/s41564-021-01014-7

This research was supported by m-CAFEs (DE-AC02-05CH11231) and the National Institute of General Medical Sciences of the National Institutes of Health (F32GM134694, F32GM131654).

Other co-authors of the paper are Alexander Crits-Christoph, Yue Clare Lou, Adair Borges, Haridha Shivram, Christine He, Michael Xu, Zeyi Zhou, Sara Smith, Rachel Rovinsky, Dylan Smock, Kimberly Tang, Netravathi Krishnappa, and Rohan Sachdeva University of California Berkeley; Trenton Owens of Berkeley Lab; and Rodolphe Barrangou of North Carolina State University.

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