Switching Off Genes with A New CRISPR Epigenetic Editing Tool

CRISPR
continues to make headlines as a powerful genomic editing tool. With a Nobel
Prize, CRISPR-based COVID-19 diagnostics, and several successful clinical
trials in the last year alone, the breakthroughs just keep coming. Now
scientists are taking CRISPR to another level – the epigenome. 

In a
recent paper published in Cell,
a team of researchers led by the Whitehead Institute has
discovered a unique way to use CRISPR to precisely control gene expression. The
new tool is called “CRISPRoff”, and it makes it possible for any gene
in a human cell to be turned off without altering the DNA sequence.

Traditional
CRISPR methods
involve editing the DNA using a protein found in bacterial immune systems along
with single guided RNA to target the specific gene of interest. The protein,
known as Cas9, acts as a molecular pair of scissors that cuts or deletes part
of the genome. This alteration is permanent and relies on the body’s own
“in-house” cellular repair mechanisms to keep the procedure limited
to just the specific change. However, potential off-target activities could
alter the function of otherwise intact genes, which is a downside of using
CRISPR.

“Though
genetic and cellular therapies are the future of medicine, there are potential
safety concerns around permanently changing the genome, which is why we’re
trying to come up with other ways to use CRISPR to treat disease,” said
Luke Gilbert, PhD, assistant professor at the University of California San
Francisco and co-senior author of the publication.

CRISPRoff
uses the same homing device part of the original CRISPR but holds back the DNA
cutting enzyme function. It can still target the gene, but it does not delete
it. Instead, it acts on the epigenome, or the proteins and chemical compounds
attached to DNA at the site of the gene. 

The
epigenome refers to the collection of all the epigenetic
marks on the DNA in a particular cell. These marks affect the way genes are
expressed and differ across tissue types, unlike DNA which is the same in every
cell. The epigenome is also dynamic in nature, and alterations can be passed
down through generations. Epigenetic mechanisms like DNA
methylation
 and histone
modifications
 play critical roles in the development of many diseases
and conditions.

For
this new tool, the scientists targeted DNA methylation in particular because it
can repress gene activity. The process of DNA methylation occurs when a small
chemical tag, known as a methyl group, gets attached to DNA. While it can occur
naturally in a cell, the CRISPRoff technique lets researchers dispense the
methylation compound directly to a site on the DNA that will then silence the
adjacent genes. 

To
construct the epigenetic editor that stimulates the DNA methylation effects,
the team developed a tiny protein mechanism that uses a single guide RNA
(sgRNA) to append a methyl group to certain spots on the DNA strand. Since this
method does not modify the genetic sequence, the process is reversible. The
researchers can use enzymes in the same way to remove the methyl group, and
they referred to this technique as “CRISPRon”.

“Now
we have a simple tool that can silence the vast majority of genes,” said
former UCSF faculty member and co-senior author Jonathan Weissman, PhD.
“We can do this for multiple genes at the same time without any DNA
damage, and in a way that can be reversed. It’s a great tool for controlling
gene expression.”

During
their testing, the team was surprised to find that their method worked on a
large majority of genes in the human genome, including regions that do not code
for protein yet control gene expression. More astounding, CRISPRoff silenced
genes in areas that did not have a high concentration of CG sequences, known as
CpG islands.

Prior
to this work, it has been believed that methylation can only silence genes at
CpG sites.  “Our work clearly shows that you don’t require a CpG
island to turn genes off by methylation. That, to me, was a major surprise, ”
said Gilbert.

To
investigate the practicality of CRISPRoff for therapeutics, the researchers
tested it in induced pluripotent stem cells, which are commonly used for
disease modeling. They found that when a gene was silenced in a treated cell,
it remained off in descendant cells as it divided. This tested true even for
maturing stem cells that normally undergo significant epigenomic modification
during transition. 

In
fact, CRISPRoff-targeted genes continued to be silenced in about 90% of the
cells that made this transition, proving that the cells kept an epigenetic
memory of the modification. The results indicate a lasting effect occurs from
just one application with CRISPRoff, making it ideal for treating rare genetic
diseases and certain types of cancer linked to genetic mutations. 

The
authors are also excited about the potential uses that this technique will
have, not only in therapeutics but for research as well. They see it as a great
tool for exploring the inner workings of the genome. As stated in their paper,
“The broad ability of CRISPRoff to initiate heritable gene silencing even
outside of CGIs expands the canonical model of methylation-based silencing and
enables diverse applications including genome-wide screens, multiplexed cell
engineering, enhancer silencing, and mechanistic exploration of epigenetic
inheritance.”

Further
research is necessary to understand the full potential that CRISPRoff presents
for therapeutics. The expectation is that this new epigenetic-type editing tool
and similar technologies will be part of the next generation of medicine. More
immediately, this new tool will allow researchers to further their
understanding of the mechanisms involved in heritability – primarily the
mystery of epigenetic inheritance.

Source: James K. Nunez, et
al. (2021). Genome-wide
programmable transcriptional memory by CRISPR-based epigenome editing
Cell.

Reference: Jason
Alvarez. New CRISPR Technology
Offers Unrivaled Control of Epigenetic Inheritance
. University
of California San Francisco, April 15, 2021.

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