03 April 2015

Editing DNA will help the staphylococcus gene

A new step in genome editing

Alexandra Bruter, <url>

American scientists have managed to slightly improve the method of genome editing – the CRISPR/Cas9 system. They found a variant of the Cas9 protein that is easier to deliver to cells than previously used variants (F. Ann Ran et al., In vivo genome editing using Staphylococcus aureus Cas9).

We talked about the CRISPR/Cas9 genome editing system in a special essay "Genome Editing: Pros and cons". Thanks to her discovery, a real revolution has taken place in genome editing over the past couple of years. Things are going so well that it is already possible to edit genes in human embryos, and a serious debate has broken out in the scientific community about whether it is ethical, safe, and necessary. So far, we have agreed that it is necessary to make sure of the safety of the method properly, and there is no direct need: in vitro fertilization with subsequent selection of embryos copes well in order to prevent the transmission of dangerous mutations by inheritance. However, in animal experiments, the method works perfectly.

The situation is quite different with the editing of the genome of individual cells of an already born organism. Considerations "against" the ethical plan arise much less often here, and if they sometimes arise, they will be neutralized by the condition and prognosis of patients with diseases that are supposed to be treated by editing the genome. Clinical trials of the use of blood stem cells with edited genomes for the treatment of HIV are already underway or will soon begin/AIDS and beta-thalassemia.

The choice of these two diseases is not accidental. Working with blood stem cells in the sense of genome modification is the easiest. You can take blood from a patient, isolate stem cells from it, multiply them, modify them, sort those where the modification actually occurred, multiply them and inject them back to the patient. The high proliferative potential of these cells and the ability to manipulate them outside the patient's body makes the question of the effectiveness of the modification procedure unprincipled.

The same is the case with the modification of the genomes of embryos that have not yet begun to divide. If you take enough embryos, there will always be one in which modification has occurred. If you do manipulations at the stage of one cell, you don't have to worry: all the cells of the grown organism will contain modification.

But so far, the method is not effective enough to modify the genome of a significant percentage of cells, for example, the liver. This is primarily due to the method of delivery of the CRISPR/Cas9 system to cells.

This system is bacterial in nature, it works as an immune system in bacteria. One of the natural enemies of bacteria is bacteriophages (viruses). In bacteriophages, the genome is most often represented by a DNA molecule. Bacteria synthesize RNA molecules that are partially complementary to the viral genome, these molecules interact with viral DNA and attract with their signal sequences the Cas9 protein, which makes a break in the DNA of the bacteriophage. Bacteria can remember enemies and, similarly to acquired immunity in humans, create archived DNA copies of antiviral RNAs in their genome. This allows you to react faster in case of a repeat attack. The study of such archived copies in bacterial genomes led to the discovery of the CRISPR/Cas9 system.

CRISPR in action (picture from the press release
Broad Institute-MIT team identifies highly efficient new Cas9 for in vivo genome editing – VM.)

If only DNA encoding the guide RNA and the Cas9 gene is injected into cells, a gap will form in the appropriate place of the DNA, which will be repaired by cell repair systems, most likely with a distortion of the DNA code. This method will be used to inactivate genes. In order for editing to occur, you also need to enter the correct version of the gene.

Today, adenoassociated viruses are considered the optimal method for the combination of efficiency and safety of introducing genetic constructs into eukaryotic cells. But they have a significant drawback for this application: the size of DNA that can be packed is noticeably limited. The limit is approximately 4,500 base pairs, while the standard Cas9 variant belonging to Streptococcus pyogenes is encoded by a gene approximately 4,200 base pairs long.

Previous attempts by the same authors to find a shorter gene in some other bacterium or to create it themselves were crowned with only partial success. But this time they found a similar gene in Staphylococcus aureus. This gene turned out to be a whole thousand base pairs shorter, and the protein was not inferior in activity to conventional variants. This made it possible to add regulatory areas to the design and generally increase the efficiency of modification.

With the help of a new protein, the authors edited the genome of liver cells containing a variant of the PCSK9 gene associated with high cholesterol levels in the blood. As a result, 40% of the liver cells in mice were modified, and the level of cholesterol in the blood decreased by almost half.

Now the authors are working to ensure that their method can be used to treat Duchenne myodystrophy – a monogenic disease, death from which inevitably overtakes carriers of the damaged gene in adolescence.

Portal "Eternal youth" http://vechnayamolodost.ru03.04.2015

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