Search and Replace

The new genome editing method turned out to be more accurate than the classic CRISPR/Cas9

Polina Loseva, N+1

Scientists from Harvard has come up with a new way to edit DNA, or rather, to rewrite it: they used viral reverse transcriptase to fit the desired sequence variant into the DNA. The primed editing method allows you to correct any type of mutations: from point substitutions to inserts or deletions. Researchers have tested it on several types of human cells and claim that it works more accurately than standard CRISPR/Cas9 and base editors. The work was published in the journal Nature (Anzalone et al., Search-and-replace genome editing without double-strand breaks or donor DNA).

The CRISPR/Cas9 molecular system is based on DNA cutting: Cas9-nuclease makes a break in both DNA chains in the right place. Then the cell can connect the ends of the gap (non-homologous connection of the ends) or insert a "patch" (homologous repair) into the place of the gap, borrowing material from the second chromosome or from the sequence provided by the experimenter. But double-strand breaks can be dangerous for the cell and cause division to stop or death, and the way to fix them is not easy to control.

In this sense, base editors are safer – this is a variant of the CRISPR/Cas system, in which the enzyme corrects only one "letter" in the DNA text, without creating double-strand breaks. However, editors are able to correct only certain types of point mutations (C→T, G→A, A→G, T→C) and are powerless against others (for example, C→A or G→T).

To expand the possibilities of fighting mutations, a group of scientists from Harvard University, led by David Liu, has developed a new strategy – primed gene editing. It does not require double-stranded breaks, and instead of the "guide" RNA that CRISPR/Cas uses to target, it includes an elongated guide RNA for primed editing (prime editing extended guide RNA, pegRNA, prgRNA). This RNA performs two functions at once: it determines the area where the editing will take place, and carries information that needs to be "written" into the gene.

The mechanism of the new system is as follows:

1. An altered Cas9 enzyme sits on the DNA strand, which unwinds the duplex into two separate chains.
2. PrgRNA is "induced" to a section of DNA, that is, it adheres to one of the strands, protecting it. On the opposite strand, Cas9 makes a single-stranded incision.
3. The other end of the prgRNA adheres to the cut thread.
4. Another enzyme, reverse trasncriptase, is attached to the Cas9 protein. It builds DNA based on prg-RNA, thereby "rewriting" the gene anew.
5. As a result, two versions of the same DNA place are obtained: the old and the new. One of them is destroyed by intracellular nucleases, and often it turns out to be the old one.
6. A new version of the DNA repair protein is "sewn" into place. At the same time, the new DNA chain and the second chain complementary to it do not match, and the repair proteins must split one of them, and build the other on the model of the opposite one. If you add another guide RNA to the system, which leads Cas9 to the "wrong" chain, then Cas9 introduces breaks into it, and repair proteins take it for an erroneous one. As a result, two copies of the desired sequence are obtained.

The authors of the new method evaluated its effectiveness on several types of human cells. According to their calculations, successful editing occurs in 20-50 percent of cases, and the frequency of erroneous inserts or deletions (indels) – about 1-10 percent. The researchers note that their system works more efficiently than the classic CRISPR/Cas9 – in similar experiments, it coped only in about 10 percent of cases. In addition, primed editing turned out to be safer: for example, in adult neurons it caused only 0.58 percent of indels, and CRISPR/Cas9 – 31 percent.

An important advantage of the new method is its versatility. By "rewriting" the gene sequence, scientists were able to eliminate any mutation – be it deletion, duplication or point replacement. They checked: the system copes with any nucleotide substitutions and other gene sequence disorders. In particular, they corrected mutations in human model cells responsible for sickle cell anemia, neurodegenerative Tay-Sachs disease and susceptibility to prion infection. The authors of the work believe that their development can be effective against 89 percent of pathogenic genetic variations that appear in the ClinVar database – all of them are small, up to 30 nucleotides, changes in the DNA sequence.

The debate about the safety of CRISPR/Cas9 and its analogues for human cells continues. In 2018, an article was withdrawn from the journal Nature Methods about the high level of inappropriate editing that the use of this system leads to. Nevertheless, this method of gene therapy is gradually moving towards clinical use. The first step in this direction was made in China, then in Recently, CRISPR-edited cells were used for the first time to fight HIV infection.

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