Editing large sections of DNA
Gene editing for the development of new treatments, the study of diseases, as well as the normal functioning of humans and animals can develop faster thanks to a new tool for cutting large DNA fragments from the cell genome. These are the results of a new study conducted at the University of California, San Francisco.
Today CRISPR is used as a research tool in laboratories around the world, and it appeared in the process of evolution in bacteria as a means of combating their ancient enemy – bacteriophage viruses. When a bacterium encounters a phage, it allows the viral DNA to embed into its own DNA, which then serves as a template for creating RNA, which in turn binds to the corresponding viral DNA in the phage itself. CRISPR enzymes then target this section of viral DNA, disable it, and kill the phage.
In his latest work on the study of such an ancient arms race, Joseph Bondi-Denomi, associate professor of Microbiology and Immunology at the University of California, together with colleagues Balint Cherge and Lina Leon, developed and tested a new CRISPR tool.
The already well-known CRISPR-Cas9 tool is similar to molecular scissors, which can be used to quickly and accurately cut out a small fragment of DNA at the target site. Then you can use different methods to insert another gene. But the new CRISPR-Cas3 system, adapted by the Bondi-Denomi group, is based on a different mechanism of the bacterial defense system. The main enzyme in this system, Cas3, is more like a molecular shredder, quickly and accurately removing much longer sections of DNA. In other words, Cas3 is much more powerful than Cas9: after detecting the target DNA, it is embedded in the chain and simply "chews" it.
The ability to remove or replace long sections of DNA on a one-time basis will allow researchers to more effectively assess the role of sections of the genome that contain DNA sequences with an as yet undefined function, which is an important factor for understanding humans and the pathogens that surround them.
Instead of doing 100 different small DNA deletions, now you can just do one deletion and see what has changed.
Because bacteria and other cell types are commonly used to produce small molecules or pharmaceutical protein preparations, CRISPR-Cas3 will allow biotech scientists to quickly and easily remove potentially pathogenic or useless DNA from these cells. CRISPR-Cas3 will also allow whole genes to be embedded in the genome for industrial, agricultural purposes or even for human gene therapy.
The researchers selected and modified the CRISPR-Cas3 system used by the bacterium Pseudomonas aeruginosa, and demonstrated in this species and in three others, including bacteria that cause diseases in humans and plants, that their more compact version successfully removes the target DNA site.
When Cas3 binds to its target DNA, it removes genes in one strand in two directions, leaving the other strand of DNA free. The deletions obtained in the researchers' experiments varied in size, in many cases including up to 100 bacterial genes. They noticed that the CRISPR-Cas3 mechanism can also simplify the replacement of a deleted part of DNA with a new sequence.
In their study, the group, by manipulating DNA sequences embedded in bacteria to restore deletions, was able to accurately establish the boundaries of these large sites for restoration, which could not be achieved with CRISPR-Cas9.
Scientists have also discovered anti-CRISPR strategies that have developed bacteriophages to fight bacteria, and they may be useful for stopping the gene editing process in humans before unwanted side effects occur or when using bacteriophages to remove unwanted bacteria.
Article by B.Csörgő et al. A compact Cascade–Cas3 system for targeted genome engineering is published in the journal Nature Methods.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on UCSF materials: CRISPR Meets Pac-Man: New DNA Cut-and-Paste Tool Enables Bigger Gene Edits.