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September 26, 2014

With Cas9 Built-In, Mouse Is CRISPR-Ready

Source: © lculig - Fotolia.com

  • The CRISPR-Cas9 gene editing system just became more convenient. The system’s Cas9 component, a nuisance to incorporate into animal models, is an intrinsic part of a newly developed mouse strain. Created by researchers at the Broad Institute and MIT, the “Cas9 mouse” is expected to simplify in vivo genome-editing experiments, particularly those in which multiple genes and cell types are to be manipulated.

    The Cas9 mouse overcomes packaging size limits. No longer will it be necessary to set aside valuable space aboard viruses or nanoparticles, the delivery vehicles of gene editing components, merely to convey the somewhat bulky Cas9 equipment. Instead, viral or nanoparticle vectors can be packed straightaway with strands of guide RNA, which direct Cas9 to DNA targets, making it less necessary to manipulate genes one at a time, or to cross animal models to produce strains possessing the desired traits.

    Details about the Cas9 mouse appeared September 25 in the journal Cell, in an article entitled, “CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.”

    The researchers described how they established a Cre-dependent Cas9 knockin mouse. They 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. And as the titled of their article suggests, the researchers used the Cas9 mouse in a modeling exercise. Specifically, they modeled lung adenocarcinoma.

    “Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma,” they wrote. “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.”

    According to a release issued by MIT, the researchers also found that cells derived from the Cas9 mouse could be extracted for use in lab experiments. The researchers 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,” said Broad core member, MIT associate professor, and paper co-author Aviv Regev, Ph.D.

    The release also indicated that the Cas9 mouse has been deposited with The Jackson Laboratory. At the Jackson Lab’s website, a page describing a Cas9 mouse strain estimated that distribution of the strain would commence December 22.

    “These CRISPR/Cas9 knockin mice have Cre recombinase-dependent expression of CRISPR associated protein 9 (cas9) endonuclease and EGFP from the mouse endogenous Gt(ROSA)26Sor locus,” the page indicated. “When used in combination with single guide RNAs and a Cre source, they allow editing of single or multiple mouse genes in vivo or ex vivo.”

    “The Cas9 mouse allows researchers to more easily perturb multiple genes in vivo,” explained Feng Zhang, Ph.D., an assistant professor at the McGovern Institute for Brain Research at MIT, core member of the Broad Institute, and 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.”

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