American researchers have developed computational models that predict the results of improving certain DNA sequences after being cut by the Cas9 protein.
As it turns out, for about one-tenth of the cases it is possible to predict with high probability that the sequence is formed there after the DNA repair system works. This allows scientists to correct a number of harmful mutations in human cells using CRISPR without using additional matrices to edit.
This writes the Chronicle .Info with a reference to hvylya.
The CRISPR-Cas9 genome editing system contains two main components – Cas9 protein and short seeds (directing RNA), which tells Cas9 where to cut the genome. This basic kit, strictly speaking, doesn't edit anything, only inserts double-strand loops in the genome in certain locations. To enter the desired sequence to this place, a third component is needed – printed DNA containing a sequence that is very necessary to be inserted into the genome. Using this matrix, the cellular system of reparations with the mechanism of homologous recombination heals the gaps in the DNA and attaches the desired pieces there.
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In the absence of a matrix for improvement (and even if there is, because homologous recombination in human cells works rather poorly), incisions are restored with the participation of other DNA repair systems, in particular, nonhomologous end connection systems (NHEJ) and final connections based on microhomology (MMEJ). ) After the operation of this system, small removal or insertion remains at the incision site, which in many cases disrupts the gene. That is why using the "basic set" of CRISPR-Cas9 is easy to break genes, but difficult to repair.
Researchers from the Massachusetts Institute of Technology decided to change the lack of a system of improvement to dignity and create a model based on machine learning, which with high probability of predicting DNA repair by the NHEJ and MMEJ mechanisms, that is, it tells what order in the cuts formed after the repair removal and insertion in at least 50 percent of cases. According to the model, it is possible to predict improvement results with an accuracy of 5-11 percent of all directing RNA for the human genome ("right-50"). To build the inDelphi model, scientists used experimental data that, after cutting the Cas9 genome, nearly two thousand sites in DNA.
After making a model, scientists experimentally confirmed its relevance – for this, from the list of RNAs directing "50" right-50 ", they chose 14, which would" regulate "Cas9 in the order of mutations (in particular, one nucleotide microdelization) certain genetic characteristics disease After repairing the gap in this place, according to inDelphi, extra nucleotides will appear. It turned out that after the CRISPR surgery and repair system, the gene sequence was restored because such microinserion averaged in 60 percent of cases.
This means that some harmful mutations (deletion or insertion) that lead to disease development can be corrected with the help of CRISPR without using a repair matrix and with sufficiently high efficiency. In total, researchers were able to take an RNA guide from the "right-50" list for 195 of these evil alleles and experimentally confirmed that those with a frequency of more than 50 percent were corrected to normal after being cut and repaired. For example, they managed to edit the mutations in the HPS1 gene in fibroblasts of patients with German-Pudlach syndrome, which caused skin pigmentation and hemophilia, as well as mutations in the ATP7A gene in Menkes disease cells.
It is also possible to edit genomes without using a matrix with help called a "basic editor" based on CRISPR-Cas, which has been able to correct all types of nucleotide substitutions. We wrote, for example, how with the help of such a tool, adult mice were cured of phenylketonuria.
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