The researchers successfully used the new “Cas9 mouse” model to edit multiple genes in a variety of cell types, and to model lung adenocarcinoma, one of the most lethal human cancers. The mouse has already been made available to the scientific community and is being used by researchers at more than a dozen institutions.
• Generation of mouse lines with Cre-dependent and constitutive Cas9 expression
• Viral/nonviral delivery of sgRNA to the brain, vasculature, immune cells, and lung
• Modeling of competition between gain- and loss-of-function mutations in lung cancer
• A convenient platform for achieving efficient genome editing in vivo
CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated KrasG12D mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.
A convenient genome-editing system
The CRISPR-Cas9 genome-editing system is one of the most convenient methods available for making these alterations in the genome. While the tool is already being used to test the effects of mutations in vitro — in cultured cell lines, for instance — it is now possible to use this tool to study gene functions using intact biological systems.
The CRISPR-Cas9 system relies on two key features to edit the genome: Cas9, a “cleaving” enzyme capable of cutting DNA; and guide RNA, a sequence that directs Cas9 to the DNA target of interest in the genome. However, the Cas9 enzyme presents some delivery challenges for in vivo applications.
“By equipping the mouse with Cas9, we relieved the burden of delivery. This frees up space for the delivery of additional elements — whether by viruses or nanoparticles — making it possible to simultaneously mutate multiple genes and even make precise changes in DNA sequences,” says Randall Platt, a graduate student at MIT working at the Broad Institute in the lab of Feng Zhang, an assistant professor at the McGovern Institute for Brain Research at MIT. Platt and Sidi Chen, a postdoc at MIT’s Koch Institute for Integrative Cancer Research working in the lab of Institute Professor Phillip Sharp, were co-first authors of the paper.
This ability to perturb multiple genes at the same time may be particularly useful in studying complex diseases, such as cancer, where mutations in more than one gene may drive disease. To demonstrate a potential application for cancer research, the authors used the “Cas9 mouse” to model lung adenocarcinoma. Previously, scientists working with animal models have had to knock out one gene at a time, or cross animal models to produce one with the needed genetic modifications, processes that are challenging and time-consuming.
“The ‘Cas9 mouse’ allows researchers to more easily perturb multiple genes in vivo,” says Zhang, who, along with Sharp, served as co-senior author of the Cell paper. “The goal in developing the mouse was to empower researchers so that they can more rapidly screen through the long list of genes that have been implicated in disease and normal biological processes.”
Researchers contributing to the paper also found that cells derived from the “Cas9 mouse” could be extracted for use in lab experiments, and were able to leverage the Cas9-expressing cells to edit immune dendritic cells even after the cells had been removed from the mouse, allowing the researchers to experiment with cells that aren’t easily accessible and often lack the shelf life to conduct such experiments.
“As we demonstrated with immune cells, the mouse allows us to experiment with cells that only remain viable for a few days ex vivo by leveraging the fact that they already express Cas9. Absent the expression of Cas9, we would not have sufficient time for the CRISPR system to work its magic,” says co-author Aviv Regev, who is an associate professor of biology at MIT. Regev’s lab, along with the lab of Broad senior associate member Nir Hacohen (a faculty member at Massachusetts General Hospital and Harvard Medical School), used the mouse to investigate dendritic cells, as reported in the Cell study.
“Genetic manipulation is one of the most critical tools we have for investigating complex circuits, and the ‘Cas9 mouse’ will help us do it more effectively,” Regev says.
The “Cas9 mouse” has been deposited with the Jackson Laboratory, in Bar Harbor, Maine, where it is available to the entire scientific community by request.
This engineered mouse should allow scientists to more easily study the dynamic genetic events that unfold during the progression of cancer and other diseases, says Luciano Marraffini, an assistant professor of bacteriology at Rockefeller University who was not part of the research team.
“This is one step up in the development of new tools based on Cas9 and CRISPR,” he says. “The development of a mouse in which Cas9 can be induced in a very specific way is something that is going to be a great tool.”
Brian Wang is a Futurist Thought Leader and a popular Science blogger with 1 million readers per month. His blog Nextbigfuture.com is ranked #1 Science News Blog. It covers many disruptive technology and trends including Space, Robotics, Artificial Intelligence, Medicine, Anti-aging Biotechnology, and Nanotechnology.
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