We fix any snips
Geneticists have learned to fix all point mutations in DNA without "scissors"
Anna Kaznadzei, N+1
Geneticists have created a genome editing tool that does not need to cut both chains of a DNA molecule and can "roll back" mutations, turning A-T nucleotide pairs into G-C pairs. Prior to that, such editors were able to change only G-C to A-T pairs, that is, they covered only half of the mutation variants. The work of Gaudelli et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage is published in the journal Nature.
Approximately half of the pathogenic mutations in human DNA are associated with the spontaneous separation of the amino group (deamination) from the cytosine nucleotide (C), which leads to the replacement of C-G pairs with T-A pairs. Deamination of adenine, in turn, leads to the appearance of inosine, which is perceived by polymerases as guanine, and this reaction could be a way to "roll back" the conversion of C-G to T-A. However, until now, enzymes capable of successfully deaminating adenines in DNA did not exist. Scientists managed to develop adenine base editors (ABES) by creating them based on adenine deaminases of tRNA "sewn" to the modified CRISPR-Cas9 system. The system in this case is necessary to find the correct DNA sequence.
The most common base editors are capable, on the contrary, of turning C-G pairs into T-A pairs. They consist of several components: a modified CRISPR-Cas9 system, which is not capable of making double-stranded incisions in DNA, but is able to find its desired site; cytidyl deaminase, capable of replacing cytosine with uracil in the five-nucleotide window of a single-stranded "bubble", which creates a Cas9 protein on DNA; and an inhibitor of uracil glycosylase, which prevents the excision of uracil and a number of other processes that affect the purity of the editing product. The nicase activity of the system also allows it to make a single-stranded incision on the "opposite", non-editable DNA chain in order to activate the work of the DNA repair system, which will replace guanine with adenine there. Such editors successfully work in the genomes of mice, plants, yeast, fish and even human embryos. They do not require DNA templates to work. You can read more about them here.
In order to create a similar editor, but turning A-T pairs into G-C, scientists from Harvard and MIT selected a number of well-known adenine deaminases of E. coli, human and mouse, which successfully deaminated free adenines, adenosines, adenosines in RNA and adenosines in paired RNA-DNA heteroduplexes. They were introduced into bacterial cells using plasmids and their activity was studied. It turned out that in its original form, none of the enzymes could effectively deaminate adenosines in double-stranded DNA.
The scientists used the methods of protein engineering and directed evolution, simulating natural selection under strictly specified conditions. They worked with bacteria that have mutations in the antibiotic resistance gene. They were treated with antibiotics, and resistance to them arose in bacteria in which genomic editing (repairing resistance genes) was successful. tRNA TadA deaminase showed the best results. To increase the efficiency of the enzyme, a number of its modifications were carried out, in particular, the selection stage showed that for successful work with DNA, it needed a mutation in the D108 position in the corresponding gene, and such a mutation was introduced into it. In addition, it turned out, among other things, that the efficiency of the enzyme increases in the case of its dimerization.
A scheme of directed evolution for obtaining effective genomic editors and introducing them into mammalian cells. Drawings from an article in Nature.
As a result, in the seventh generation, scientists received effective editors (ABE7.10), replacing the necessary A-T pairs with G-C pairs with an efficiency of up to 50 percent. At the same time, the level of side insertions and deletions was, on average, no more than 0.1 percent. More common methods of genomic editing based on CRISPR-Cas9 systems are associated with the introduction of double breaks in the DNA chain, and this process, as a rule, generates much more side inserts and deletions. In addition, editing with ABEs turned out to be more accurate, since his work produced fewer non-target substitutions.
Working scheme of the ABE editor
Scientists separately conducted an experiment demonstrating the effectiveness of ABE in editing pathogenic mutations. Mutations in the beta-globin gene are known to cause a wide range of blood diseases. Some of their carriers, however, are resistant to them due to mutations in the promoter regions of gamma globin genes. Scientists have developed a specific ABE editor that introduces mutations in these areas, replacing A-T with G-C. It demonstrated 29- and 30-percent efficacy on two promoters in HEK293T cells.
A similar experiment was conducted with the HFE gene, a mutation in which causes hemochromatosis in humans. The enzyme successfully worked in 28 percent of cases, replacing the 845th nucleotide in the gene, and, accordingly, changing the amino acid of the corresponding protein from tyrosine to cysteine.
Scientists note that the conversion of cytosine into thymine or uracil occurs from 100 to 500 times a day in each human cell. This process can lead to mutations in important parts of DNA and lead to the emergence of a variety of genetic diseases. Obtaining genomic editors capable of restoring the original DNA sequence, both in the case of A-T and G-C pairs, is an important step for genetic engineering methods.
And you can find out about the first experiments with human embryos and editors who turn C-G pairs into T-A pairs here.
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