MIT: Genome-wide studies of mouse livers using the gene-editing system CRISPR

The liver's ability to regenerate itself is amazing
.
Even if more than 70% of the organs are removed, the remaining tissue can regenerate a complete new liver
.

Kristin Knouse, an assistant professor of biology at MIT, wants to find out how the liver achieves this regeneration and hopes to understand how to induce other organs to do the same
.
To that end, her lab has developed a new method to conduct genome-wide studies
of mouse livers using the gene-editing system CRISPR.

With this new technique, researchers can study how each gene in a mouse's
genome affects a particular disease or behavior.
In a paper describing the technique, the researchers identified several genes important for liver cell survival and proliferation that had not previously been found in studies of cells grown in laboratory dishes
.

"If we really want to understand mammalian physiology and disease, we should study the processes of these living organisms wherever possible, because this is where we can study biology in their most native environment"
.

Knouse is the senior author of the new paper, published today in
the journal Cell Genomics.
Heather Keys, director of the Functional Genomics Platform at the Whitehead Institute, is a co-author of the study
.

Extracellular environment

As a graduate student at MIT, Knouse used regenerated liver tissue as a model to study one aspect of cell division, chromosome separation
.
In this study, she observed that liver cells divide differently
than liver cells in lab dishes.

"What I learned from this study is the extent to which cell division, something intrinsic to the cell, is clearly influenced by the extracellular environment, and we've always thought that cell division has nothing to do with
anything outside the cell," she said.
"When we study cells in culture, we lose the influence of
the extracellular environment.
"

However, many types of research, including genome-wide screening using techniques such as CRISPR, are more difficult to deploy
at the scale of whole organisms.
The CRISPR gene-editing system consists of an enzyme called Cas9, which cuts DNA
at a given location under the guidance of a strand of RNA called guide RNA.
This allowed the researchers to knock out a gene
in each cell in a large number of cells.

While this approach can reveal genes and proteins involved in specific cellular processes, it has proven difficult
to efficiently deliver CRISPR components into enough cells in the body to make them available for animal studies.
In some studies, researchers have used CRISPR to knock out about 100 genes of interest, which is useful if they know which genes they want to study, but this limited approach doesn't reveal new genes
associated with specific functions or diseases.

Some research groups have used CRISPR to perform genome-wide screening in brain and skin cells, but these studies require large numbers of mice to spot significant impacts
.

"For us, and I think many other researchers, the limited experimental manageability of mouse models has long hampered our ability to
delve into mammalian physiology and disease issues in an unbiased and comprehensive manner," Knouse said.
"That's what I really want to change, to bring experimental adaptations that were once limited to cell culture into organisms, so that we're no longer limited to exploring the fundamentals of physiology and disease
.
"

To guide strands of RNA into liver cells, the dominant cell type in the liver, Knus decided to use lentivirus, an engineered non-pathogenic virus commonly used to insert genetic material into a cell's genome
.
She injected the guide RNA into newborn mice so that once the guide RNA was integrated into the genome, it was passed on to the offspring of liver cells
as the mice grew.
After months of laboratory effort, she was able to consistently express directed RNA in tens of millions of liver cells, enough to perform genome-wide screening
in a single animal.

Cellular health

To test the system, the researchers decided to look for genes that influence liver cell fitness — their ability to
survive and proliferate.
To do this, they provided a library of more than 70,000 guide RNAs, targeting more than 13,000 genes, and then determined the effect
of each knockout on cellular fitness.

The mice used for the study were engineered so that Cas9 could be turned on
at any point in their lives.
The researchers used a group of 4 mice—2 males and 2 females—to initiate Cas9 expression
when the mice were 5 days old.
After three weeks, the researchers screened their liver cells and measured the amount
of each guide RNA.
If a particular guide RNA is abundant, it means that its target gene can be eliminated without causing fatal harm
to the cell.
If the guide RNA does not appear on the screen, it means that knocking out the gene is fatal to the cell
.

This screening yielded hundreds of genes involved in hepatocellular fitness, and the results were very consistent
across the four mice.
The researchers also compared
the genes they found to genes associated with human liver disease.
They found that genetic mutations in neonatal liver failure syndrome also led to the death
of liver cells in screening.

The screening also revealed key health genes
not found in studies of liver cells cultured in laboratory dishes.
Many of these genes are involved in interactions
with immune cells or extracellular matrix molecules.
These pathways may not have been present in the screening of cultured cells because they involve the cell's interaction with the external environment, Knouse said
.

By comparing the results from male and female mice, the researchers also identified several genes that had sex-specific effects on health, which were not possible by studying cells alone
.

Update and regenerate

Knouse now plans to use the system to identify genes
that are essential for liver regeneration.

"Many tissues, such as the heart, cannot regenerate because they lack stem cells and differentiated cells cannot divide
.
However, the liver is also a highly differentiated tissue that lacks stem cells, but it retains this amazing ability to regenerate itself after injury," she said
.
"Importantly, the genome of liver cells is no different from
the genome of heart cells.
All these cells have the same operating manual in their nucleus, but the liver cells are apparently reading different sentences
in the manual in order to regenerate.
What we don't know is, what are these sentences? What are these genes? If we can identify these genes, perhaps one day we can direct heart regeneration
.
”

This new screening technique could also be used to study diseases
such as fatty liver disease and cirrhosis.
Knus's lab is also working to extend this approach to other organs
beyond the liver.

"We need to find ways
to efficiently guide RNA into other organizations," she says.
"By overcoming this technical hurdle, we can build the same experimental adaptations
we now have for liver, heart, or other problems.
"

The research was supported by the NIH Director's Early Independence Award from the National Institutes of Health, a National Cancer Institute Koch Institute Support (Core) grant, and the Scott Cook and Sig Osterby Fund
.