Saturday, December 13, 2014

New way to turn genes on discovered: Technique allows rapid, large-scale studies of gene function (CRISPR)



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.
In new research, scientists have shown that they can reliably turn on any gene of their choosing in living cells.

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.

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Biotechnology Department

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