Us scientists have synchronised the system CRISPR-Cas9 with the help of light.
Photosensitive chemical modification guide RNA have allowed not only at the same time
to cut DNA in a population of cells, but also to make it much faster than usual. A study published in Science, the magazine came out and related editorial.
The efficiency of genome editing using CRISPR-Cas9 is affected by many
factors, and one of them — the formation of molecular complexes. On
to complex RNA-Cas9
recognize the target, contacted her and carried out the incision, it takes time. In some
cells it is faster, in others slower, or not happening.
The consequence of this, in particular, can be a problem of mosaic embryos, in
which different cells of the future body contain different options
gene. Even in the same cell different copies of the gene on two chromosomes can
to cut the module with different speed and efficiency. Most often it happens that
one copy of the gene is already cut and “sewn shut”, and another set do not
got. For this reason, editing of diploid cells is certain
To synchronize the work of CRISPR, researchers are developing
induced options system that can be activated by a signal from the outside. Biophysics
from Johns Hopkins University in Baltimore under the leadership of Taggia Ha (Taekjip Ha) has developed a system
photoinductive cutting the CRISPR-Cas9 system,
based on chemical modification guide RNAS. After stimulation with light
a wavelength of 365 or 405 nanometers, the chemical group blocking
complementary binding with DNA in a complex with Cas9, tiny and allow you to make
the incision in the whole population of cells simultaneously.
Search target and cut DNA in the CRISPR system consists of
multiple stages. First, the complex of Cas9 and guide RNA (source in bacteria it is composed of two
molecules — crRNA and tracrRNA)
looking for a PAM sequence,
which consists of only three “letters”, and binds to it. Then double
the DNA helix rasplatitsya and plot guide RNA that actually “directs”
the nuclease to the target, mates with one of the chains by the principle of complementarity
next to PAM. For cutting
need pair plot of 20 nucleotides, but in order to complex
recognize the target, pairing it up enough the first half (12 nucleotides),
the area adjacent to the PAM. If the site
the guide RNA is artificially trimmed to this length, the entire complex will be stuck
DNA, and will sit there, nothing cutting.