New CRISPR/Cas9 gene knock-in system to aid liver cancer modeling Genome Medicine。

Title: CRISPR-SONIC: targeted somatic oncogene knock-in enable rapids in vivo cancer modeling
Journal:
Haiwei Mou†, Deniz M. Ozata†, Jordan L. Smith†, Ankur Sheel, Suet-Yan Kwan, So Hren Hren, Al Kukuku, Zachary Kennedy, Yueying Cao and Wen Xue
Published: 2019/04/16
Digital ID: 10.1186/s13073-019-0627-9
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Recently published on the open access journal
, A new method for modeling mice with liver cancer has been reported, using the CRISPR/Cas9 system to quickly tap cancer-related genes into the DNA of mice.
"To better understand tumor biology, conduct preclinical research, and find potential treatment strategies for patients, we need effective tumor models," said Wen Wang, co-author of the study and director of the RNA Therapy Institute at the University of Massachusetts School of Medicine. Existing methods or methods for tapping into cancer-causing genes to model cancer are inefficient or difficult to control the number of copies knocked in and in. CRISPR/Cas9 makes it possible to insert large pieces of DNA into specific locations in the genome, which we call the target gene base, and to apply them to human cells in the laboratory as well as in mice. We have developed a new system, CRISPR-SONIC, that provides flexible gene tapping in mouse models of liver cancer with high accuracy.To address the existing problems of cancer modeling and meet the need for rapid and effective animal modeling, Mo Haiwei, Deniz Ozata, and Jordan L. Smith developed this new system that uses crispr/Cas9 gene editing systems to insert cancer-causing genes into the genomes of living mice. The CRISPR/Cas9 system consists of a guide RNA and Cas9 enzyme. Wizard RNA is a short sequence of nucleotides that attach to a specific target DNA sequence in the genome. Since the wizard RNA is also connected to Cas9 enzymes, it can direct Cas9 to the target DNA sequence. Cas9 then cuts the DNA, removes a single nucleotide/entire gene, or inserts the nucleotide/entire gene during DNA repair.
the authors performed a three-step operation in this study using CRISPR/Cas9 with 2 wizard RNA. First, one of the wizards, RNA, and Cas9 enzymes work together to cut the target DNA location. In the second step, another wizard, RNA and Cas9, will cut a DNA ring, or prosury supply, and the third step will insert a lithic ring that has been cut into a line into the target position.
to test this method, the authors used it to insert a green fluorescent protein (GFP) gene into laboratory-cultured mouse cells. After successfully entering the cell's DNA, the gene produces a green fluorescent protein that is visible under a laser, indicating that the gene was successfully inserted and expressed. After successful testing in laboratory cells, the authors tested the method in mice.
, a researcher at the university, said: "We observed that about 10 per cent of the liver cells in our sample successfully took GFP with using CRISPR-SONIC. This is a huge improvement over the knock-in efficiency of about 0.5% of those previous methods.
the authors then tested the CRISPR-SONIC system, which includes guide RNA, Cas9 enzymes, and a carcinogenic gene granules, to see if it could be used to model mice with the second-highest incidence of intrapatulphic bile tube cancer in liver cancer.
The most common genetic mutations that cause this type of cancer occur in the anti-cancer gene TP53 (about 26-44% of all cases) and the cancer-causing gene KRAS (about 16-18% of all cases)," said Mu Haiwei. Past studies have shown that if the two mutations occur together, they can cause intra-liver bile tube cancer in mouse models. We knocked KRAS with CRISPR-SONIC and added another guide RNA to knock out the cancer-suppressing gene TP53. This is important in cancer modeling because KRAS cannot cause tumor formation in the presence of p53. A
the mice were injected with CRISPR-SONIC, the authors observed tumors forming in the mice's livers. The control mice did not develop tumors after being injected with guide RNA, Cas9, and a luminescent DNA fragment, rather than a cancer-causing gene.
. Smith said, "We tested our method with RAS cancer genes, but we think any carcinogenic gene fragment can be customized in this way." We showed how CRISPR-SONIC can be used to model liver cancer, but this approach also has the potential to be applied to other tissues and organs. The
also showed that this method could also be used to build bioluminescent cancer models that allow researchers to monitor the growth and development of cancer cells in real time.。 CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3′-UTR of mouse β-actin, allowing co-expression of reporter genes or oncogenes from the β-actin promoter. We showed that knock-in of oncogenic Ras and loss of p53 efficiently induced intrahepatic cholangiocarcinoma in mice. Further, our strategy can generate bioluminescent liver cancer to facilitate tumor imaging. This method simplifies in vivo gain-of-function genetics by facilitating targeted integration of oncogenes.
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(Source: Science.com)