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

The two new methods can simultaneously perform CRISPR editing on genes of multiple cell types

So far, CRISPR enzymes have been used to edit the genome of one cell at a time: for example, they cut, delete, or add genes to specific cells in tissues or organs or a microorganism growing 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 multiple different species at the same time.

Although this technology is still only used in laboratory environments, it can be used to edit and track microbes in natural communities, such as in the intestines or roots of plants, where hundreds or thousands of different microbes gather together

If there is no way to track gene insertions (in this case, barcodes are used), the inserted genes may disappear anywhere because the genes are usually shared between microorganisms

In order to successfully edit the genes of multiple members of a microbial community, scientists at the University of California, Berkeley must develop two new methods: Environmental Transformation Sequencing (ET-Seq), which allows them to evaluate the editability of specific microorganisms; And DNA-editing all-in-one RNA-guided CRISPR-Cas transposase (DART), which allows highly specific targeted DNA to be inserted into the genome location determined by the guide RNA

Benjamin Rubin, a postdoctoral researcher at the University of California, Berkeley, said: "Breaking and changing the DNA in isolated microorganisms is essential to understanding the role of DNA

The ability of the "shotgun" to edit multiple types of cells or microorganisms at the same time may be useful in current industrial-scale systems.

Postdoctoral fellow Brady Kleis said: “Ultimately, we may be able to eliminate the genes that cause gut bacteria to get sick, or make plants more efficient by modifying their microbial partners

Rubin and Cress are both in the laboratory of CRISPR-Cas9 inventor Jennifer Doudna, and Spencer Diamond, a project scientist at the Institute of Innovative Genomics (IGI), is the co-first author of the paper describing the technology, which was published today (December) 6th) in the journal Nature Microbiology

From census to editing

Diamond works in Jill Banfield's laboratory.

Metagenomic sequencing technology has made tremendous progress in the past 15 years

But he likened it to a census: it provides unparalleled information about which microbes are present in what proportions and what functions these microbes can perform in the community

He said: "There is an idea of ​​metabolic transmission, that is, no single microorganism performs a series of huge metabolic functions, but in most cases, each individual organism only performs a single step of a process, so there must be The delivery of some metabolites

The research team is led by Banfield, Professor of Earth and Planetary Sciences, Environmental Science, Policy and Management at the University of California, Berkeley, and Jennifer Dudner, Professor of Molecular and Cell Biology and Chemistry at the University of California, Berkeley, and a researcher at the Howard Hughes Medical Institute , Won the 2020 Nobel Prize in Chemistry for the invention of CRISPR-Cas9 genome editing technology

The team first developed a method to determine which microorganisms in the community are actually susceptible to gene editing

Then, Cress developed a targeted delivery system called DNA editing all-in-in-RNA-guided CRISPR Cas transposase (DART), which uses a CRISPR-Cas enzyme similar to CRISPR-cas9 to target specific DNA sequence and insert barcode for transposition
.

In order to test the DART technology with a more realistic microbial community, the researchers took a stool sample from a baby and cultured it to create a stable community composed mainly of 14 different types of microorganisms
.
They were able to edit individual E.
coli strains in the population to target disease-related genes
.

Researchers hope to use this technology to understand artificial, simple communities, such as plants and their associated microbial communities in a closed box
.
They can then manipulate the community genes in this closed system and track their effects on barcoded microorganisms
.
These experiments are part of a 10-year project called m-cafe funded by the Department of Energy to understand the response of a simple grassland microbial community to changes in the outside world
.
m-cafe is a project of soil microbial community analysis and function evaluation
.
Banfield, Doudna and Deutschbauer are part of the m-cafe project
.

references:

“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, 6 December 2021,  Nature Microbiology .