CRISPR-Cas, which does not cut DNA

CRISPR-Cas9 was used for the first time to "turn on" genes in a living organism without changing the genome

Anna Kerman, XX2 century, based on the Los Angeles Times: Scientists use CRISPR to turn genes on without editing their DNA

The revolutionary CRISPR-Cas9 technique initially gained fame as a way to accurately change nucleotide sequences in DNA strands. Thanks to CRISPR-Cas9, scientists have been able to edit genomes with an accuracy previously unavailable.

Now the researchers have demonstrated another area of application of the technique: changing gene expression without interfering with the genome itself. For the first time, the staff of the Salk Institute for Biological Studies managed to "turn on" useful genes in the body of living mice suffering from muscular dystrophy, type 1 diabetes mellitus and acute renal failure. As reported in an article published in Cell, the health status of experimental animals improved in more than 50% of cases (In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation).

Earlier studies have shown that CRISPR can be used to alter gene expression in cell cultures. However, the possibility of such an application of the technique in living organisms has been demonstrated for the first time.

"We are one step closer to treating people," said Hsin–Kai Liao, a postdoc at the Salk Institute and the first co-author of the paper.

Alexis Komor, a biochemist at the University of California, San Diego, who was not involved in the new project, agrees: "This is a really neat in vivo study that has begun to build a bridge between using CRISPR-based techniques to change the genomes of cells in Petri dishes and using they are used in clinical practice."

CRISPR-Cas9 is often called the "Swiss army knife" for gene editing, because this technique is capable of performing several operations at once. Firstly, it works like a "find" button in your text editor. CRISPR is easy to set up to search for specific sequences of letters in DNA. When the letters are found, the CRISPR-Cas9 complex "turns" into molecular scissors – an enzyme (usually Cas9) attaches to the selected site and "cuts"
 DNA. And to replace a "broken" gene with a working one, scientists attach a DNA chain to CRISPR, hoping that the cell will "decide" to use the new DNA to repair the broken chain. This often works, but, unfortunately, not always, which means that the use of CRISPR-Cas9 can lead to unnecessary deletions and insertions in the genetic code.

To overcome the shortcomings of the method, the scientists decided to experiment with a modified version of CRISPR-Cas9, designed to "turn on" and "turn off" certain genes. In this version of CRISPR, the Cas9 enzyme is deactivated. It is still able to attach to DNA, but not "cut" it. Instead, the new Cas9 "glues" molecules to the DNA chain indicating the need for activation of the selected gene.

The new version of CRISPR-Cas9 is interesting not only because it protects the genome from the occurrence of random mutations. The changes resulting from the work of the "new CRISPR" are reversible and easy to correct. In addition, when using modified CRISPR-Cas9, a process is actually reproduced that "includes" genes in vivo.

Scientists have been trying to use CRISPR in living organisms since 2013. However, so far, success in this field has not been achieved. According to the researchers, the problem was in creating a working CRISPR-Cas9 delivery system.

Viruses are usually used to introduce new materials into the cells of animal organisms. But such a delivery system has its own limitations, it only works with relatively small molecules. The instructions that scientists used to activate certain genes in cell culture were too large to carry them with viruses.

To overcome this limitation, the researchers separated the Cas9 enzyme from the instructions. Now the enzyme was injected into the cell with the help of one virus, and the instructions with the help of another.

It took about four years of trial and error, but eventually the scientists came to success. For the first time, the results were recorded on an experimental mouse with weakened muscles. She was "turned on" the genes that ensure the growth of muscle tissue.

Skeletal muscle mass (top) and laminin fiber size (bottom) in the treated mouse (right) compared to the control (left). Figure from the press release of Salk scientists modify CRISPR to epigenetically treat diabetes, kidney disease, muscular dystrophy – VM.

In the following months, a group of researchers used the new system to treat mice with other diseases, in particular, with acute renal failure and type 1 diabetes mellitus.

It will take a long time before the new technique will be used to treat people. Now scientists are planning to find out how effectively their development will function in the organisms of larger animals. In addition, thorough safety studies of the new method are needed.

The authors hope that over time, the "new CRISPR" will begin to be used in clinical practice. However, the researchers emphasize, the end result may be the treatment of diseases, rather than complete healing. Even if everything goes according to an optimistic scenario, patients will need regularly repeated courses of gene therapy.

The researchers also note that theoretically the new technique can be used to treat many diseases, including Alzheimer's disease, as well as to combat the aging process in general.

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