In new research, scientists have shown that they can reliably turn on any gene of their choosing in living cells.
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.
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.
Monday, December 8, 2014
A new National Science Foundation-supported project will provide computational tools designed to help identify and characterize the gene diversity of the residents of these microbial communities. The project, being done by researchers at the Georgia Institute of Technology and Michigan State University, will allow clinicians and scientists to compare the genomic information of organisms they encounter against the growing volumes of data provided by the world's scientific community.
The tools will be hosted on a web server designed to be used by researchers who may not have training in the latest bioinformatics techniques. A prototype system containing a limited number of computational tools is already available at http://enve-omics.ce.gatech.edu and is attracting more than 500 users each month.
"Across many areas of science, we are dealing with communities of microorganisms, and one challenge we've had is to identify them because we haven't had good tools to tell apart individual microbes from the mixtures," said Kostas Konstantinidis, an associate professor in the School of Civil and Environmental Engineering at Georgia Tech and the project's principal investigator. "Our tools will be designed to deal with the genomes of whole communities of organisms."
Current techniques identify individual microbes by examining their small subunit ribosomal RNA (SSU rRNA) genes, but the new tools will allow scientists to analyze entire genomes and meta-genomes.
"With the dawn of the genomic era, we can now get the whole genome of these organisms to see not only the ribosomal RNA, but also all the genes in the genome to get a better understanding of what the each organism's potential might be," said Konstantinidis. "There will be many advantages for looking at all the genes instead of just one, the SSU rRNA, such as to identify which organisms encode toxins or the enzymes for breaking down pollutants."
The ability to identify and enumerate the organisms in complex communities using culture-independent, genomic technologies and associated bioinformatics algorithms is becoming more important as scientists study organisms that can't be grown in the lab. The majority of the world's organisms resist traditional lab culture, meaning they have to be studied in the field and identified through genetic information.
Konstantinidis and his research group are studying such communities in the water of lakes in Chattahoochee River system in Georgia and elsewhere. They are examining how these communities respond to perturbations, such as oil or pesticide spills, and the role that different members of the community play in breaking down pollutants.
"These tools actually come from our research practice," said Konstantinidis. "We came to the point where we couldn't process the data to answer the questions we wanted to ask. That led us to this new project to develop the tools we and others need to interrogate the data and get the information we are looking for."
A single liter of lake water may contain as many as 500 different species, and together, their genomic information can total tens of billions of gene-coding letters. From Lake Lanier alone, the team has generated 200 gigabytes of genomic data.
Among the challenges ahead is building an infrastructure able to handle the growing amounts of genomic information produced worldwide.
"We will have to develop some computational solutions for the problems of keeping up with all the new data becoming available," said Konstantinidis. "We need to make tools that have high throughput to keep up with data volumes that are increasing geometrically."