Over the past decade, the CRISPR-Cas9 gene editing system has revolutionized genetic engineering, allowing scientists to make targeted changes to organisms' DNA. While the system could potentially be useful in treating a variety of diseases, CRISPR-Cas9 editing involves cutting DNA strands, leading to permanent changes to the cell's genetic material. Now, in a paper published online in Cell on April 9, researchers describe a new gene editing technology called CRISPRoff that allows researchers to control gene expression with high specificity while leaving the sequence of the DNA unchanged. Designed by Whitehead Institute Member Jonathan Weissman, University of California San Francisco assistant professor Luke Gilbert, Weissman lab postdoc James Nuñez and collaborators, the method is stable enough to be inherited through hundreds of cell divisions, and is also fully reversible.
The classic CRISPR-Cas9 system uses a DNA-cutting protein called Cas9 found in bacterial immune systems. The system can be targeted to specific genes in human cells using a single guide RNA, where the Cas9 proteins create tiny breaks in the DNA strand. Then the cell's existing repair machinery patches up the holes.Because these methods alter the underlying DNA sequence, they are permanent. Plus, their reliance on "in-house" cellular repair mechanisms means it is hard to limit the outcome to a single desired change.That's where the researchers saw an opportunity for a different kind of gene editor—one that didn't alter the DNA sequences themselves, but changed the way they were read in the cell. This sort of modification is what scientists call "epigenetic"—genes may be silenced or activated based on chemical changes to the DNA strand. Problems with a cell's epigenetics are responsible for many human diseases such as Fragile X syndrome and various cancers, and can be passed down through generations.
Epigenetic gene silencing often works through methylation—the addition of chemical tags to to certain places in the DNA strand—which causes the DNA to become inaccessible to RNA polymerase, the enzyme which reads the genetic information in the DNA sequence into messenger RNA transcripts, which can ultimately be the blueprints for proteins. To build an epigenetic editor that could mimic natural DNA methylation, the researchers created a tiny protein machine that, guided by small RNAs, can tack methyl groups onto specific spots on the strand. These methylated genes are then "silenced," or turned off, hence the name CRISPRoff. Because the method does not alter the sequence of the DNA strand, the researchers can reverse the silencing effect using enzymes that remove methyl groups, a method they called CRISPRon.
As they tested CRISPRoff in different conditions, the researchers discovered a few interesting features of the new system. For one thing, they could target the method to the vast majority of genes in the human genome—and it worked not just for the genes themselves, but also for other regions of DNA that control gene expression but do not code for proteins. "That was a huge shock even for us, because we thought it was only going to be applicable for a subset of genes," says first author Nuñez. Also, surprisingly to the researchers, CRISPRoff was even able to silence genes that did not have large methylated regions called CpG islands, which had previously been thought necessary to any DNA methylation mechanism.
Read: https://phys.org/news/2021-04-reversible-crispr-method-gene-underlying.html
The classic CRISPR-Cas9 system uses a DNA-cutting protein called Cas9 found in bacterial immune systems. The system can be targeted to specific genes in human cells using a single guide RNA, where the Cas9 proteins create tiny breaks in the DNA strand. Then the cell's existing repair machinery patches up the holes.Because these methods alter the underlying DNA sequence, they are permanent. Plus, their reliance on "in-house" cellular repair mechanisms means it is hard to limit the outcome to a single desired change.That's where the researchers saw an opportunity for a different kind of gene editor—one that didn't alter the DNA sequences themselves, but changed the way they were read in the cell. This sort of modification is what scientists call "epigenetic"—genes may be silenced or activated based on chemical changes to the DNA strand. Problems with a cell's epigenetics are responsible for many human diseases such as Fragile X syndrome and various cancers, and can be passed down through generations.
Epigenetic gene silencing often works through methylation—the addition of chemical tags to to certain places in the DNA strand—which causes the DNA to become inaccessible to RNA polymerase, the enzyme which reads the genetic information in the DNA sequence into messenger RNA transcripts, which can ultimately be the blueprints for proteins. To build an epigenetic editor that could mimic natural DNA methylation, the researchers created a tiny protein machine that, guided by small RNAs, can tack methyl groups onto specific spots on the strand. These methylated genes are then "silenced," or turned off, hence the name CRISPRoff. Because the method does not alter the sequence of the DNA strand, the researchers can reverse the silencing effect using enzymes that remove methyl groups, a method they called CRISPRon.
As they tested CRISPRoff in different conditions, the researchers discovered a few interesting features of the new system. For one thing, they could target the method to the vast majority of genes in the human genome—and it worked not just for the genes themselves, but also for other regions of DNA that control gene expression but do not code for proteins. "That was a huge shock even for us, because we thought it was only going to be applicable for a subset of genes," says first author Nuñez. Also, surprisingly to the researchers, CRISPRoff was even able to silence genes that did not have large methylated regions called CpG islands, which had previously been thought necessary to any DNA methylation mechanism.
Read: https://phys.org/news/2021-04-reversible-crispr-method-gene-underlying.html
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