A new method, which uses the CRISPR/Cas9 system to generate liver cancer mouse models by rapidly adding (knocking in) cancer genes to DNA, is being reported in the open access journal Genome Medicine.
Wen Xue of the RNA Therapeutics Institute at University of Massachusetts Medical School, the corresponding author said: "Well-defined tumor models are needed to better understand cancer biology, perform preclinical studies and identify potential therapeutic strategies for patients. Existing methods used to make models of cancer by adding (knocking in) cancer-causing oncogenes have low efficiency, or it can be difficult to control where the gene is added and how many copies are made. CRISPR/Cas9 allows for the integration of large DNA sequences into a specific place on the genome, called a target locus, and is applicable for human cells in the lab or in mice. We have developed a new system - CRISPR-SONIC - which allows a greater degree of precision for flexible gene knock-in in live cancer mouse models."
To tackle existing issues with cancer modelling and to satisfy the need for rapid and efficient generation of live models, the new system, developed by Haiwei Mou, Deniz Ozata and Jordan L. Smith, uses the CRISPR/Cas9 gene editing system to insert cancer-causing oncogenes into the genomes of live mice. The CRISPR/Cas9 system consists of a guide RNA and the Cas9 enzyme. Guide RNAs are short sequences of nucleotides (DNA building blocks) that attach to a specific target DNA sequence in a genome. As the guide RNA also attaches to the Cas9 enzyme, it can be used to guide Cas9 to a target sequence. Cas9 then cuts the DNA, allowing for single nucleotides or whole genes to be removed or inserted as the DNA is repaired.
The authors used CRISPR/Cas9 with two guide RNAs in a three-step process. First, one of the guide RNAs and the Cas9 enzyme cut the target DNA locus. Second, the other guide RNA and Cas9 cut and linearize a round piece of DNA (called a plasmid donor), which in a third step is inserted into the DNA at the target locus.
To test their approach, the authors used the method to add a green fluorescent reporter gene (GFP) into mouse cells cultured in the laboratory. This gene, when added to the DNA of a cell, produces a green fluorescent protein which is visible under a laser, indicating that the insertion has worked and the gene is being expressed. After testing the method in cells in the laboratory, the authors tested the method in mice.
Deniz Ozata said: "We observed that, following our use of CRISPR-SONIC, approximately 10% of the liver cells in our sample were GFP positive. This is a significant improvement compared to the 0.5% knock-in efficiency of previous methods."
Next, the authors tested if they could use their CRISPR-SONIC system - comprising the targeted guide RNAs, the Cas9 enzyme and a common oncogene plasmid - to model intrahepatic cholangiocarcinoma (ICC), the second most common liver cancer, in live mice.
Haiwei Mou said: "The top mutations that drive this cancer are of the tumour suppressor gene TP53 (26-44% of cases) and the oncogene KRAS (16-18%). It has previously been shown that if the two mutations occur together, they drive ICC in mouse models. We used CRISPR-SONIC to knock in KRAS, while also adding a guide-RNA that would target and disable (knock out) the TP53 tumour suppressor gene. This is important for cancer modelling, because KRAS is not associated with tumour formation in the presence of p53."
One month after injection, the authors observed tumour formation in the livers of treated mice. Control mice injected with the guide RNA, Cas9 and a luminescent DNA sequence, rather than an oncogene, did not develop tumours.
Jordan L. Smith said: "We tested our strategy on RAS oncogenes but we assume that any desired oncogenic DNA sequence could be used and tailored to model other cancer types. While we show the use of CRISPR-SONIC in creating models of liver cancer, the method may also potentially be applied to other tissues and organs."
The authors also showed that the method can be used to create bioluminescent cancer models which allow researchers to monitor how cancer grows and develops in real time.
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Notes to editor:
1. Research article:
CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in vivo cancer modeling
Mou et al. Genome Medicine 2019
For an embargoed copy of the research article, please contact Anne Korn at BMC.
After the embargo lifts, the article will be available here: https://genomemedicine.biomedcentral.com/articles/10.1186/s13073-019-0627-9
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