20 November 2019

The art of cutting and sewing

Sewing and cutting DNA have become more accurate

Vera Mukhina, "Trinity Variant"

A lot has been said about CRISPR/Cas technology – a kind of universal genomic scissors - in recent years. This technology has really revolutionized and allowed us to significantly expand the possibilities of molecular biology. The method made it possible to edit almost any DNA in the laboratory without much effort: cut it in the right places and, if necessary, replace individual sections with others, resulting in a genetically modified organism with the desired properties.

In theory, this technology could help in the future not only to create genetically modified animals and plants, but also to help people with genetic diseases. Attempts are already underway, but the results have not yet been published. The technique has been tested on patients with sickle cell anemia, beta-thalassemia and esophageal cancer; in addition, a single case of editing human embryos is known (in China). This case caused a wave of indignation and controversy in scientific circles: the experiment was put "on the knee" and – judging by the scraps of leaked information – failed.

CRISPR/Cas technology is far from perfect. The method does not always work correctly, and especially often failures occur in human cells. Experiments on cell lines suggest that correct editing occurs only in 3-20% of cases.

The main problems begin at the stage of "sewing up" DNA. CRISPR/Cas easily manages to make an incision in a given place, but then the method relies on the fact that the cell itself will "patch up" this incision according to the sample provided by scientists. Depending on the nature of the incision, the cell uses different repair systems, differing in accuracy. CRISPR/Cas cuts both strands of DNA, and the sloppiest repair system is responsible for such repairs.

Two years ago, a team of researchers led by biochemist David Liu, head of the a laboratory at the private Broad Research Institute (Cambridge, Massachusetts) and professors at Harvard University came up with a variant of CRISPR/Cas, which allows you not to cut both DNA strands, but to cut one of the two and repair this incision yourself. The main enzyme that carried out all the operations consisted of two pieces: a mutant Cas9 protein, capable of cutting one DNA chain in the right place, and an enzyme that carefully modifies the "letters" of DNA, were sewn together. Unfortunately, the new method had an obvious limitation – it could replace only one "letter" of the DNA code, and larger changes were impossible.

Recently, the authors of this method published an article (Anzalone et al., Search-and-replace genome editing without double-strand breaks or donor DNA // Nature 2019), in which they proposed an improved version of the method: now it is possible to carefully change any number of "letters" in a row. The trick is to use a new hybrid enzyme. As in the previous case, one of its halves consists of a mutant Cas9 protein. The second part consists of reverse transcriptase, an enzyme that can synthesize DNA from an RNA matrix.

The whole work of the new editor can be divided into several stages (Fig. 1).


The algorithm of the new genome editor.

  1. The genomic editor finds the desired section of DNA and makes an incision at a given point.
  2. On one side of the incision, the chain is built up in the "correct" sequence.
  3. The old piece of DNA is replaced by a new sequence.
  4. On the second DNA chain – previously intact (intact) – an incision is made opposite the modified piece.
  5. The cellular DNA repair system recognizes it and repairs it according to the pattern of the already corrected chain.

The work of the new editor requires not only an enzyme – a machine for cutting and synthesizing DNA – but also a template, on the model of which these changes will be carried out. Its role is a single RNA molecule containing sequences complementary to the original and required sequence, as well as a site that binds to the enzyme. The classical CRISPR/Cas system also needs samples of the source and result sequences, but there they are arranged a little differently and lie on different molecules.

Due to its similarity, RNA can find a section of DNA that needs to be changed and bind to it, forming an RNA-DNA complex. In this form, an enzyme finds it, binds and makes an incision in the DNA.

DNA synthesis enzymes do not know how to do it "just like that", for work they need the tail of an already existing molecule, which they can complete, and a sample – a complementary chain. The free DNA tail appears due to the incision, and as a sample, the enzyme uses a piece of RNA with the changes made. Thus, the sequence of the RNA molecule depends entirely on where and what changes will be made, and the enzyme is a universal performer.

It is important that both the template and the enzyme are initially located close to the edited site, are promptly included in the synthesis and control it. This reduces the likelihood of interference by other random cellular enzymes and increases the predictability of the result.

As a result of the hybrid enzyme, the edited piece looks like this. One chain still remains intact, and the second is notched and completed so that the piece being changed has two versions – the old and the new. They partially coincide, so that the complementary chain can interact with both.

The next task of the researchers is to make sure that the introduced piece of the sequence is fixed in the DNA: it replaces the old sequence on the same chain and changes the second complementary chain for itself. In part, the solution of these tasks is entrusted to the cell's own resources, but it still does not take its course, but is kept under control.

DNA repair systems see a single-stranded DNA break and seek to repair it, but they have two options (marked with asterisks in Fig. 2): with the restoration of the desired sequence (in the figure on the left) or with the addition of a new one (on the right).


The author's drawing.

In the direction of the new sequence, the cell is quickly inclined by an enzyme called FEN1 endonuclease, which hunts for freely dangling DNA tails and destroys them. The DNA strand has a direction, and the endonuclease is able to bite off only from one end of it. Scientists have calculated that this end will be an old piece of DNA. After the old piece of DNA has been eaten by endonuclease, the cellular repair system has no choice – and it sews up the gap with the inclusion of a new sequence.

The second step, the correction of the complementary chain, also takes place with the help of cellular DNA repair systems under the supervision of researchers. The new section of DNA is not completely complementary to the second chain, and the cell is trying to fix this too. She faces a new choice: which of the chains to take as a sample – edited or untouched? In order to mislead the cell and force it to correct the chain with the source code, scientists go to a trick. Using the same mutant Cas9 enzyme and an RNA sample, they make a new incision, this time in an intact chain. This dramatically affects the cellular choice: she regards this incision as a source of error and corrects it following the example of the whole chain – with an already edited section.

Despite the external complexity of the manipulations, this method, according to its authors, is much neater and more effective than the previous one: depending on the cell lines on which the method was tested and on the size of the insert, the number of successfully edited sections varied from 20 to 50%, and the number of errors ranged from 10%.

The authors of the study tested the new method on different types of inserts. In total, they performed more than 175 "operations" to correct the genomes of cell lines, among which there were both single-letter and larger inserts, substitutions and deletions within a few dozen letters. According to them, this method can correct approximately 89% of all known DNA variants associated with genetic diseases.

Of course, the new method has yet to be comprehensively tested by other research groups, but already now it looks very promising. Unlike conventional CRISPR/Cas, it uses cellular repair systems to a minimum, and if it uses them, it takes only neat ones and controls their operation.

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