American researchers have developed a computer model that predicts the outcome of repairing a particular DNA sequence after it was lowered by Cas9 protein.
As it turns out, about a dozen cases can be predicted with a high likelihood that is created there after the operation of the DNA repair system. This allowed scientists to correct numerous harmful mutations in human cells using CRISPR without the use of a matrix for editing.
This is what Chronicle says. Info with regard to hvilia.
The CRISPR-Cas9 genome editing system contains two major components – Cas9 protein and short semen (rectal RNA), which tells Cas9 where the genome is reduced. This basic set, strictly speaking, does not regulate anything, it simply inserts the double gap in the genome in a particular location. In order to insert the desired sequence to this site, a third component is required – a DNA template that contains the sequence itself to be inserted into the genome. Using this matrix, the cellular repair system by the homologous recombination mechanism treats the emptiness in the DNA and incorporates the desired part there.
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In the absence of a repair matrix (and even if it exists, since homologous recombination in human cells is rather poor), the cut is regenerated with the involvement of other DNA repair systems, in particular the system of non-hormonal endpoints (NHEJs) and the ultimate connection based on microhomology MMEJ). After the operation of these systems, small deletions or inserts remain in place of the cut, which in most cases disturbs the gene. Therefore, the use of the "basic set" of CRISPR-Cas9 is easy to break the gene, but it is difficult to repair it.
Researchers from the Massachusetts Institute of Technology have decided to achieve a lack of repair systems in dignity and create a model based on machine learning, which is highly probable to predict the outcome of DNA repair using the NHEJ and MMEJ mechanisms, i.e. It talks about which sequence in the cut is formed after the repair by erasing and inserting in at least 50 percent of cases. According to the model, it is possible to predict the outcome of reparation with such an accuracy of 5-11 percent of all rectal RNA for the human genome ("precise-50"). In order to create an inDelphi model, scientists used experimental data that, after cutting the Cas9 genome, almost two thousand places in the DNA.
After modeling, scientists experimentally confirmed its relevance – for this, from the "precise-50" list by directing RNA, they chose 14, which would "place" Cas9 in the sequence with a mutation (especially a nucleotide microdeletia) characteristic of a particular genetic disease. After removing the emptiness at this site, according to InDelphi, an additional nucleotide should appear. It turned out that after the work of CRISPR and the repair system, the gene sequence was restored due to such microinsertion on average in 60 percent of cases.
This means that some harmful mutations (deletions or insertions) that lead to the development of the disease can be corrected by CRISPR without the use of a correction matrix and sufficiently high efficiency. In total, the researchers were able to collect RNA's from the "precision-50" list for 195 such malicious alleles and experimentally confirmed that those with a frequency of more than 50 percent were corrected normally after cutting and repair. For example, they succeeded in regulating the mutation in the HPS1 gene in patients with fibroblasts with Pudlach's syndrome, leading to a reduction in skin pigmentation and haemophilia, as well as mutations in the ATP7A gene in Menkes' disease cells.
It is also possible to change the genome without the use of a matrix using the so-called "basic editors" based on CRISPR-Cas, which can already correct all types of nucleotide substitutions. We wrote, for example, that with the help of such a tool, adult mice were healed by phenylketonuria.
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