American researchers have developed a computational model that provides for the outcome of the repair of a particular DNA sequence after being cut from the Cas9 protein.
As it turned out, for about a tenth of the cases it is possible to predict with high probability what sequence is formed there after the work of DNA repair systems. This allowed scientists to correct a number of harmful mutations in human cells using CRISPR without the additional use of the modification matrix.
This writes the Chronicle. Info with reference to hvylya.
The CRISPR-Cas9 genome editing system contains two main components: the Cas9 protein and a short seed (direction RNA), which tells Cas9 where to cut the genome. This basic kit, in the strict sense, does not change anything: it simply inserts a double-stranded slit in the genome in a certain position. To insert the desired sequence in this place, a third component is needed: the DNA of the model containing the sequence to be inserted into the genome. Using this matrix, the cellular repair system using the homologous recombination mechanism heals the gap in the DNA and incorporates the desired piece there.
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In the absence of a repair matrix (and even if it exists, since homologous recombination in human cells works quite badly), the incision is restored with the participation of other DNA repair systems, in particular the system of non-homologous terminal connections (NHEJ) and end connections based on mycology (MMEJ). After the operation of these systems, small deletions or insertions remain at the incision site, which in most cases disrupts the gene. That's why using the "basic set" of CRISPR-Cas9 is easy to break a gene, but difficult to solve.
Researchers at the Massachusetts Institute of Technology have decided to transform the lack of repair systems into dignity and have created a model based on machine learning, which with a high probability predicts the result of DNA repair through mechanisms NHEJ and MMEJ, that is, indicates which sequence in the cut is formed after the repair with deletions and insertions in at least 50% of the cases. According to the model, it is possible to predict the outcome of the repair with such accuracy for 5-11% of all the direct RNAs for the human genome ("precise-50"). To build an inDelphi model, scientists used experimental data that, after cutting the Cas9 genome, saw nearly two thousand sites in the DNA.
After creating the model, scientists have experimentally confirmed its relevance – for this, from the list of "precision-50" ANNs, they chose 14, which would "set" Cas9 on a sequence with a mutation (in particular, a microdeletion of a nucleotide) characteristic of a particular genetic disease. After repairing the vacuum in this place, according to inDelphi, an extra nucleotide should appear. It was discovered that after CRISPR and the repair system functioned, the genetic sequence was restored on average due to such micro-insertion in 60% of cases.
This means that some harmful mutations (deletions or insertions) that lead to the development of diseases can be corrected with the help of CRISPR without using a repair matrix and with a sufficiently high efficiency. In total, the researchers were able to collect RNA guides from the list of "precise-50" for 195 such harmful alleles and experimentally confirmed that with a frequency above 50% they are corrected to normal after the cut and the repair. For example, they managed to modify a mutation in the HPS1 gene in fibroblasts from patients with German-Pudlach syndrome, leading to skin pigmentation and hemophilia, as well as a mutation in the ATP7A gene in Menkes disease cells.
It is also possible to modify the genome without using a matrix with the help of the so-called "basic editors" based on CRISPR-Cas, which can already correct all types of nucleotide substitutions. We wrote, for example, how with the help of such an instrument adult mice were treated by phenylketonuria.
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