Up to the nucleotide: details

Genome editing has reached point mutations

Alexander Ershov, N+1

Biologists from Harvard University have modified the genome editing method using the CRISPR/Cas9 system in such a way that now point changes in DNA can be introduced without cutting its strands, which can be dangerous in itself. The new technique involves correcting point mutations in a much more direct way than has been done so far. The work was published in the journal Nature (Komor et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage).

Previously, the genome editing scheme, both in the case of point mutations and when making large inserts or deletions, was the same. Scientists injected an RNA guide sequence into the cell, which was supposed to find the target gene, as well as a scissor enzyme capable of cutting DNA in the place where the guide sequence would indicate.

The appearance of a rupture stimulates the cells to get rid of it as quickly as possible. If, after the rupture, the cellular enzymes do not have a suitable sample "at hand" to restore the whole sequence, then a simple fastening of the broken ends of the DNA strand occurs (the non-homologous end joining pathway). This way is dangerous because enzymes can accidentally introduce extra bases, knocking down the amino acid frame of the gene reading.

If the task of researchers is simply to "break" a gene, such carelessness of enzymes works in favor of scientists. But if the goal is to pointwise replace one nucleotide with another (as in the case of many hereditary diseases), or to introduce a new sequence, then it is necessary to supply cells with the correct sequence-a sample. Such a sequence, if it is sufficiently similar to a broken one, replaces it with itself – this process is called homologous recombination. By simultaneously supplying cells with a scissor enzyme with a guide RNA and a sample sequence with the desired mutation, point or any other changes can be introduced into the genome.

The problem is that the actual recombination efficiency is low (no more than ten percent) and for point mutations is highly redundant and overcomplicated. Therefore, the authors of the article have invented a new approach to introducing point mutations. It, like the classic CRISPR/Cas9, at the first stage involves the search for a genomic target due to the guide RNA, but no gap is introduced into it. Instead, one nucleotide is directly replaced by another using a special enzyme attached to Cas9.

In their work, the authors cite such a scheme. One of the nucleotides of the GC pair is first deprived of the amine group – cytosine turns into uracil (C→U). An incorrect GU pair is formed. Such pairs can be found by a point repair system (this is a separate cellular system that is not related to the repair of breaks), and one of the nucleotides can be cut out. If G is cut out of the "unpaired pair", and not U, then in the end we get a gene where a new AU (or AT, in the genetic sense, this is the same thing) appeared in place of the original GC pair.

Scheme for replacing a pair of GC nucleotides with AT from an article in Nature

Of the two bases of the "unpaired pair", one intended for removal is selected by enzymes randomly, unless we are talking about freshly synthesized DNA. However, scientists were able to shift the randomness in the right direction by making single-stranded breaks in only one of the two DNA chains due to the residual activity of Cas9. As a result, scientists have succeeded in replacing the sequence in 15-75 percent of cells in the culture with the desired one. At the same time, the error rate (when a break in the DNA did occur and it ended with a simple DNA bonding) was less than one percent of cases.

The main disadvantage of the new method is that for each type of change that needs to be introduced into DNA, you need to choose your own set of enzymes. The new method so far allows you to change only CG pairs to AT. In order to introduce a reverse mutation, it is required to use other enzymes and, possibly, a different approach. Nevertheless, it potentially allows the treatment of many genetic diseases, a very large proportion of which is associated with just such point mutations. You can read more about the CRISPR/Cas9 method here.

Portal "Eternal youth" http://vechnayamolodost.ru  21.04.2016