Using a gene-editing system originally
developed to delete specific genes, researchers have now shown that they can
reliably turn on any gene of their choosing in living cells. The findings are
expected to help researchers refine and further engineer the tool to accelerate
genomic research and bring the technology closer to use in the treatment of
human genetic disease. Using a gene-editing system originally developed to
delete specific genes, MIT researchers have now shown that they can reliably
turn on any gene of their choosing in living cells.
This new application for the
CRISPR/Cas9 gene-editing system should allow scientists to more easily determine
the function of individual genes. This approach also enables rapid functional
screens of the entire genome, allowing scientists to identify genes involved in
particular diseases.
A new function for CRISPR
The CRISPR system relies on cellular machinery
that bacteria use to defend themselves from viral infection. Researchers have
previously harnessed this cellular system to create gene-editing complexes that
include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that
is programmed to bind to a specific genome sequence, telling Cas9 where to make
its cut.
Scientists have tried
to do this before using proteins that are individually engineered to target DNA
at specific sites. However, these proteins are difficult to work with. There
have also been attempts to use CRISPR to turn on genes by inactivating the part
of the Cas9 enzyme that cuts DNA and linking Cas9 to pieces of proteins called
activation domains. These domains recruit the
cellular machinery necessary to begin reading copying RNA from DNA, a process
known as transcription.
However, these efforts have been
unable to consistently turn on gene transcription. In previous efforts,
scientists had tried to attach the activation domains to either end of the Cas9
protein, with limited success. From their structural studies, the MIT team
realized that two small loops of the RNA guide poke out from the Cas9 complex
and could be better points of attachment because they allow the activation
domains to have more flexibility in recruiting transcription machinery.
Using their revamped system, the
researchers activated about a dozen genes that had proven difficult or
impossible to turn on using the previous generation of Cas9 activators. Each
gene showed at least a twofold boost in transcription, and for many genes, the
researchers found multiple orders of magnitude increase in activation.
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In new
research, scientists have shown that they can reliably turn on any gene of
their choosing in living cells.
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Genome-scale activation screening
Once the researchers had shown that
the system was effective at activating genes, they created a library of 70,290
guide RNAs targeting all of the more than 20,000 genes in the human genome.
They screened this library to identify
genes that confer resistance to a melanoma drug called PLX-4720. This drug
works Drugs of this type work well in patients whose melanoma cells have a
mutation in the BRAF gene, but cancer cells that survive the treatment can grow
into new tumors, allowing the cancer to recur.
To discover the genes that help cells
become resistant, the researchers delivered CRISPR components to a large
population of melanoma cells grown in the lab, with each cell receiving a
different guide RNA targeting a different gene. After treating the cells with
PLX-4720, they identified several genes that helped the cells to survive --
some previously known to be involved in drug resistance, as well as several
novel targets.
Studies like this could help
researchers discover new cancer drugs that prevent tumors from becoming
resistant.
Scientists have tried to do
large-scale screens like this by delivering single genes carried by viruses,
but that does not work with all genes.
Posted By;-
Biotechnology Department
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