“Community Genetic Editing” – Using CRISPR to Modify Genes in Multiple Cell Types Simultaneously



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

To date, CRISPR enzymes have been used to alter the genomes of one type of cell at a time: they cut, remove, or add genes to a specific type of cell in a tissue or organ, for example, or to a type of microbe growing in a test tube.

Now the University of California, Berkeley, the group that invented CRISPR-Cas9 genome editing technology almost 10 years ago has found a way to add or change genes within a community of many different species simultaneously, opening the door to what one might call “community edition”.

Although this technology is still exclusively applied in the laboratory, it could be used both to modify and to track modified microbes within a natural community, such as in the gut or on the roots of a plant where they congregate. hundreds or thousands of different microbes. Such monitoring becomes necessary as scientists talk about genetically modifying microbial populations: inserting genes into microbes in the gut to solve digestive problems, for example, or modifying the microbial environment of crops to make them more resistant to diseases. pests.

Without a way to track gene insertions – using a barcode, in this case – those inserted genes could end up anywhere, as microbes regularly share genes with each other.

Explanation of ET-Seq and DART

To successfully edit genes within multiple members of a microbial community, scientists at UC Berkeley had to develop two new methods: Environmental Transformation Sequencing (ET-Seq), at the top, which enabled them assess the editability of specific microbes; and the all-in-one DNA-editing CRISPR-Cas transposase (DART), which enabled highly specific, targeted DNA insertion into a genome location defined by a guide RNA. The DART system is bar-coded and compatible with ET-Seq, so when used together, scientists can insert, track and assess the efficiency and specificity of the insertion. Credit: Jill Banfield Lab, UC Berkeley

“Break and change DNA within isolated microorganisms has been essential to understanding what this DNA does, ”said Benjamin Rubin, postdoctoral researcher at UC Berkeley. “This work helps bring this fundamental approach to microbial communities, which are much more representative of how these microbes live and function in nature.”

While the ability to modify many types of cells or microbes at once could be useful in current industry-wide systems – bioreactors for bulk cell culture, for example, the most common application. more immediate can be a tool for understanding the structure of complex communities of bacteria, archaea and fungi, and the flow of genes within these diverse populations.

“Eventually, we may be able to eliminate the genes that cause disease in your gut bacteria or make plants more efficient by creating their microbial partners,” said postdoctoral fellow Brady Cress. “But probably, before we do that, this approach will give us a better understanding of how microbes work in a community.”

Rubin and Cress – both in the lab 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 technique. published today (December 6). ) in the review Natural microbiology.

From census to publication

Diamond works in the laboratory of Jill Banfield, a pioneering geomicrobiologist in the field of community sequencing, or metagenomics: shotgun sequencing of all the DNA of a complex community of microbes and assembly of this DNA into the complete genomes of all. these organisms, some of which probably have never been seen before and many of which are impossible to grow in a laboratory dish.

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

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

“There is this idea of ​​metabolic transfers – that no individual microbe performs a huge chain of metabolic functions, but for the most part each individual organism performs only one step in a process, and that there has to be a certain transfer of metabolites between organisms, “he said.” That’s the hypothesis, but how do we actually prove it? How do we get to a point where we don’t just look at birds anymore, we can actually do a little manipulation and see what happens? That was the genesis of the community assembly.

The research team was led by Banfield, professor of Earth and Planetary Sciences at UC Berkeley and of Environmental Science, Policy and Management, and Jennifer Doudna, Professor of Molecular and Cellular Biology and Chemistry at UC Berkeley, Howard Hughes Medical Institute Fellow and Co-Laureate. of the 2020 Nobel Prize in Chemistry for the CRISPR-Cas9 genome editing invention.

The team first developed an approach to determine which microbes in a community are actually susceptible to gene editing. The screening technique developed by Rubin and Diamond, called ET-seq (environmental transformation sequencing), uses as a probe a transposon, or jumping gene, which easily fits randomly into many microbial genomes. By sequencing the DNA of the community before and after the introduction of the transposon, they were able to identify the species of microbes capable of incorporating the transposon gene. The approach was based on techniques developed by co-author Adam Deutschbauer at the Lawrence Berkeley National Laboratory. In an experiment involving a community of nine different microbes, they managed to insert the same transposon into five of them using different transformation methods.

Cress went on to develop a targeted delivery system called DNA-editing All-in-one. RNA-Guided CRISPR Cas Transposase (DART) which uses a CRISPR-Cas enzyme similar to CRISPR-Cas9 to focus on a specific DNA sequence and insert a barcode 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 individually E. coli strains within this community, targeting genes that have been associated with the disease.

The researchers hope to use the technique to understand artificial and simple communities, such as a plant and its associated microbiome, in a closed box. They can then manipulate the genes of the community within this closed system and track the effect on their barcode microbes. These experiments are one aspect of a 10-year program funded by the Ministry of Energy called m-CAFEs, for Microbial Community Analysis and Functional Evaluation in Soils, which seeks to understand the response of a simple grass microbiome 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” By 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, Natural microbiology.
DOI: 10.1038 / s41564-021-01014-7

The 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).

The other co-authors of the article 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 of UC Berkeley; Trenton Owens of the Berkeley lab; and Rodolphe Barrangou from North Carolina State University.


Previous Do I have to apply for a instant approval credit card, even if I don't need one?
Next How Rolling Loud DJ Five Venoms continued to make money during the pandemic