What do you know about the new CRISPR Cas9 gene editing system?

Reported in Genome Medicine, a new CRISPR Cas9 gene editing system has been developed that adds genes to create mouse models of liver cancer.

A new method, which uses the CRISPR Cas9 gene editing system to generate mouse models of liver cancer by rapidly adding cancer genes to DNA.

CRISPR Cas9 gene editing technology

Wen Xue of the RNA Therapeutics Institute of the Faculty of Medicine at the University of Massachusetts said, "Well-defined tumor models are needed to better understand cancer biology, conduct preclinical studies, and identify potential therapeutic strategies for patients.

"Existing methods used to create cancer models by adding (injecting) cancer-causing oncogenes have low efficacy, or it can be difficult to control where the gene is added and how many copies are made.

"CRISPR Cas9 allows the integration of large DNA sequences into a specific genome location, called the target locus, and applies to human cells in the laboratory or in the mouse. We have developed a new system, CRISPR-SONIC, which allows an increased degree of precision for the flexible introduction of genes into living cancer mouse models. "

To tackle the problems associated with cancer modeling and the need for fast and efficient generation of live models, the new system, developed by Haiwei Mou, Deniz Ozata and Jordan L. Smith, utilizes the system. Cas9 CRISPR gene editing to insert carcinogens. oncogenes in the genome of living mice. The CRISPR / Cas9 system is composed of a guide RNA and the Cas9 enzyme.

Guide RNAs are short nucleotide sequences (DNA building blocks) that bind to a specific target DNA sequence of 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 DNA, allowing single nucleotides or whole genes to be deleted or inserted during DNA repair.

Using the Cas9 CRISPR Gene Edition

The authors used CRISPR Cas9 with two guide RNAs in a three-step process. First, one of the guide RNAs and Cas9 enzyme cut the locus of the target DNA. Second, the other guide, RNA and Cas9, cut and linearize a round piece of DNA (called a plasmid donor) that, in a third step, is inserted into the DNA at level of the target locus.

To test their approach, the authors used the method to add a green fluorescent reporter gene (GFP) in mouse cells grown in the laboratory. This gene, added to the DNA of a cell, produces a green fluorescent protein visible to the laser, indicating that the insertion has worked well and that the gene is being expressed. After testing the method in the laboratory cells, the authors tested the method in mice.

Ozata said, "We observed that after using CRISPR-SONIC, about 10% of the liver cells in our sample were positive for PFM. This is a significant improvement over the knock-in efficiency of 0.5% of the previous methods. "

Next, the authors tested whether they could use their CRISPR-SONIC system, composed of targeted guide RNAs, Cas9 enzyme and a common oncogenic plasmid, to model intrahepatic cholangiocarcinoma (ICC). , the second most common liver cancer in living mice. .

Observe the formation of tumors

Haiwei Mou said, "The main mutations at the origin of this cancer are the TP53 tumor suppressor gene (26-44% of cases) and the KRAS oncogene (16-18%). It has already been shown that if both mutations occur together, they induce ICC control in mouse models. We used CRISPR-SONIC to inoculate KRAS, while adding a guide RNA that would target and inhibit (inactivate) the TP53 tumor suppressor gene.

"This is important for cancer modeling because KRAS is not associated with tumor formation in the presence of p53."

One month after injection, the authors observed the formation of tumors in the liver of treated mice. Control mice injected with the guide RNA, Cas9 and a luminescent DNA sequence, rather than an oncogene, did not develop tumors.

Smith said, "We have tested our strategy on oncogenes RAS, but we assume that any desired oncogenic DNA sequence could be used and adapted to modeling other types of cancer.

"Although we show the use of CRISPR-SONIC in the creation of liver cancer models, the method can also potentially be applied to other tissues and organs."

The authors also showed that this method could be used to create bioluminescent cancer models allowing researchers to track the evolution and progression of cancer in real time.