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Broad Scientists Create Complete Knock-out Screen of Mouse with CRISPR/Cas9

NEW YORK (GenomeWeb) – Scientists have used CRISPR/Cas9 to create a comprehensive genetic screen of the mouse genome. Using guide RNAs developed to knock out genes in the 2.6 billion base pair mouse genome, the scientists revealed new genes involved in tumor evolution and metastasis.

The scientists, led by Feng Zhang and Phillip Sharp of the Broad Institute, published their results today in Cell, noting that their work is the first complete genome screen developed for in vivo studies.

"Genome-scale guide RNA libraries are a powerful screening system, and we're excited to start applying it to study gene function in animal models," Zhang said in a statement. "This study represents a first step toward using Cas9 to identify important genes in cancer and other complex diseases in vivo."

In the study, in vitro cells from a mouse model of non-small cell lung cancer that doesn't usually metastasize were transduced by lentiviral vectors packing the CRISPR/Cas9 gene-editing nuclease and the Broad Institute's "mouse genome-scale CRISPR knockout library A." This library of 67,405 guide RNAs allows the nucleases to target 20,611 protein-coding genes and 1,175 microRNA precursors in the mouse genome; using it with the in vitro cells ensured that only a single gene was knocked out in each cell and that each gene was knocked out in at least several hundred cells. The researchers then transplanted the cohort of about 30 million cells into mice and found that they caused highly metastatic tumors that spread to the lungs.

Next-generation sequencing helped the scientists to identify which genes had been knocked-out in both the primary tumors as well as in the metastases, indicating that the genes are likely tumor suppressors that normally inhibit growth and metastasis.

The results highlighted some well-known tumor suppressor genes in human cancer, including Pten, Cdkn2a, and Nf2, but included some genes not previously linked to cancer. The screen also implicated several microRNAs in tumor evolution and metastasis.

The system is a breakthrough in reverse genetics, allowing scientists to completely knock out genes at the DNA level and look for phenotypic changes at the organism level. Previous methods of whole-genome screens involved RNA interference "knock downs," which do not effect changes at the DNA level. And Broad Institute scientists had previously performed genome-wide screens using CRISPR-Cas9 technology in cellular models, but that approach does not capture the complexity of a whole organism.

"Tumor evolution is an extremely complex set of processes, or hallmarks, controlled by networks of genes," Sharp, a co-senior author of the paper, said in a statement. For cancer to metastasize, malignant cells must leave the primary tumor, enter blood vessels to travel to a distant site in the body, leave the blood vessels, and thrive in a new environment. Sharp said that the in vivo application of gene editing offers a way to investigate each step in tumor evolution and identify the genes that regulate those hallmarks of cancer.

The authors wrote that their study provides a roadmap for in vivo Cas9 screens, noting that future studies can take advantage of this model to explore other oncogenotypes, delivery methods, or metastases in other organs.