Technical advances in DNA sequencing and genomics have given scientists access to troves of information about genetic changes in tumors.
But the functional consequences of specific mutations—permanently turning on a communication pathway in cells that drive them to proliferate, for example—have proven difficult to study. Previous tools to make targeted changes to genomes have been, until now, too expensive and cumbersome for widespread practical use, explained David Baltimore, Ph.D., of the California Institute of Technology, and his colleagues in a recent perspectiveExit Disclaimer in Science. But now, a technique for gene editing, borrowed from bacteria, has changed that, the authors said.
From Bacteria to Breakthrough
Known as CRISPR-Cas9, this technology has led to a breakthrough in genomic engineering. Unlike earlier tools for genome editing, such as zinc-finger nucleases and transcription activator-like effector nucleases (TALENs), the technology makes it much easier and faster for cancer researchers to study mutations identified by The Cancer Genome Atlas and test new therapeutic targets.
You can parallel what CRISPR has done for genome editing with what microarrays have done for gene expression—it’s a vast improvement on the speed and throughput of the technology.
Dr. Ji Luo
“The primary advantage of CRISPR is its ability to easily edit the genome in a precise fashion. You can parallel what CRISPR has done for genome editing with what microarrays have done for gene expression—it’s a vast improvement on the speed and throughput of the technology, said Ji Luo, Ph.D., of NCI’s Laboratory of Cancer Biology and Genetics.
Lou Staudt, M.D., Ph.D., of NCI’s Center for Cancer Research, called CRISPR-Cas9 a transformative technology that could yield insights into how the cancer cells are wired. “We know that mutated genes form abnormal regulatory networks within the cells, and some aspects of those molecular networks can be just as essential as the gene that is mutated,” he said. “Those regulatory networks can give you new targets for therapy. The promise of this technology is to expand the targets in cancer therapy beyond just those genes that are mutated.”
Jennifer Doudna, Ph.D., of the University of California, Berkeley, and Emmanuelle Charpentier, Ph.D., of Hannover Medical School, Germany, and Umeå University, Sweden, discovered CRISPR when they were studying bacterial immunity.
Bacteria use special sequences in their genome, called clustered regularly interspaced short palindromic repeats (CRISPR), to guide a DNA-cutting enzyme called Cas9 to specific sequences of DNA. Bacteria use this system to recognize and destroy foreign DNA, such as attacking viruses. But the system—a DNA-cutting enzyme guided by an RNA molecule to a specific sequence—is exactly the tool needed to make sequence-specific edits in the DNA of a human cell.
CRISPR in Action
Dr. Luo's laboratory at CCR is using CRISPR to identify potential cancer drug targets. To do this, his lab is constructing a customized library of small-guide RNAs (sgRNAs) that guide the Cas9 enzyme to target some 9,000 potentially druggable genes in the human and mouse genome. With this library of sgRNAs, Dr. Luo's laboratory can efficiently conduct high-throughput screens in cancer cell lines. The entire library is delivered into a cancer cell line using a virus vector, and each cell is allowed to only take up one sgRNA. The result is a population of cells each with a unique gene that has been knocked out.
To identify genes that are important to the growth and survival of cancer cells, the researchers look for changes in the frequency of sgRNAs in the population as the cells grow and divide. If a gene that promotes or limits growth is knocked out, the cells carrying the corresponding sgRNA will grow at a different rate than other cells, Dr. Luo explained. Using next-generation DNA sequencing, the researchers in his lab can keep track of the frequencies of thousands of sgRNAs in the population as the cells grow and divide, effectively running thousands of tests in parallel, all in one experiment.
“By deep sequencing all the cells in the population, we can quickly identify changes in the relative abundance of each sgRNA in a very complex library over time, and infer the function (e.g., growth, cell division, cell death) of their target genes,” explained Dr. Luo.
Dr. Staudt is using genome-wide libraries of sgRNAs to identify genes required for the proliferation and survival of lymphoma cells. He explained that although many of these screens were possible with a previous technology called RNA interference (RNAi), the CRISPR method isn’t plagued by as many off-target effects—that is, inadvertent targeting of genes other than the one of interest—and, unlike RNAi, CRISPR completely eliminates expression of the target gene. The result is data that are cleaner and of higher quality, he said.
Moreover, whereas RNAi can be used only to inhibit gene expression, CRISPR can be used to enhance the expression of existing genes to study their effect on cellular function and test their ability to drive cancer growth, explained Dr. Staudt. Using CRISPR to control the expression of genes hypothesized to drive cancer growth allows researchers to differentiate between the mutations that are driving cancer growth from those that are innocuous “passenger” mutations, he explained. And comparing the behavior of cancer and normal cells with the same CRISPR-generated mutation can help researchers identify gene targets that cancer cells depend on for survival but that normal cells can do without.
Researchers can also use CRISPR to introduce specific mutations to see how many—and in what order—are needed to turn a normal cell into a cancer cell. “Being able to recapitulate the process of oncogenic transformation in [cell culture] in a defined way teaches us a lot about how cancers evolve,” explained Dr. Luo. These types of studies can also help researchers to understand how cancer cells came to become addicted to their driver oncogene, and ultimately help identifying possible therapeutic approaches.
Another use of these CRISPR libraries is to identify potential combination therapies that will inhibit the evolution of drug resistance in tumors, Dr. Luo added. “The question is: If we have a drug which we know works well, how can we find other genes to target to improve response rate and prevent the rise of drug resistant tumors?”
To address the first question, one approach is to treat cancer cells with a low, sublethal dose of the drug and look for which genes, when knocked out by CRISPR, would sensitize the cells to this lower dose of the drug, he said. Such genes could therefore serve as a potential co-target of the drug in future combination therapy. To address the drug resistance question, a cancer cell line is treated with a high concentration of the drug, and researchers can investigate which genes, when knocked out by CRISPR, are rendering the cells resistant to the drug. “This doesn’t necessarily lead to a therapy, but it does lead to an understanding of the development of drug resistance, which could help the development of second line treatments.” said Dr. Luo.
There is a big opportunity to deploy the CRISPR technology in cancers growing in living animals, said Dr. Staudt. “Cancer phenotypes don’t just relate to what the cancer cells do; they also have to do with how the tumor interacts with the microenvironment and the immune system,” he explained. Studying the real-time behavior of tumors in the environment of an animal’s body hasn’t been done yet, but it is conceivable using CRISPR, Dr. Staudt said.
CRISPR, like any technology, has limitations. Issues that need to be considered include the possibility of off-target genetic alterations associated with this approach, as well as unintended consequences of on-target alterations. Little is known about the physiology of cells and tissues that have undergone genome editing and there is evidence that complete loss of a gene could lead to compensatory adaptation in cells over time, said Dr. Luo.
Biology experiences revolution every time a new technology comes along. CRISPR is one of those technological revolutions.
Dr. Ji Luo
Even though CRISPR’s clinical applications are many years away, this technology is already bearing fruit in the lab. “Historically, biology has always been propelled by the availability of new technologies to allow you to answer questions that were not previously answerable,” said Dr. Luo. “Biology experiences revolution every time a new technology comes along. CRISPR is one of those technological revolutions.”
ver historia personal en: www.cerasale.com.ar [dado de baja por la Cancillería Argentina por temas políticos, propio de la censura que rige en nuestro medio]//
weblog.maimonides.edu/farmacia/archives/UM_Informe_Autoevaluacion_FyB.pdf - //
weblog.maimonides.edu/farmacia/archives/0216_Admin_FarmEcon.pdf - //
www.proz.com/kudoz/english_to_spanish/art_literary/523942-key_factors.html - 65k - // www.llave.connmed.com.ar/portalnoticias_vernoticia.php?codigonoticia=17715 // www.frusculleda.com.ar/homepage/espanol/activities_teaching.htm // http://www.on24.com.ar/nota.aspx?idNot=36331 ||