Chinese bioengineering experts have developed a molecular tool called smACGmax, which can simultaneously replace three types of nucleotides within a specific DNA segment. This technology is designed for targeted gene evolution, enabling the creation of cells with new beneficial properties and accelerating the development of pharmaceuticals.

Principles of Directed Evolution

Directed in situ evolution is a laboratory simulation of natural selection. During this process, numerous random mutations are introduced into a target gene, after which the most successful variants are selected. This approach is essential for rapidly creating proteins and cells with new characteristics, such as resistance to diseases or toxins. In nature, such changes might take millions of years to occur—or might never happen at all. Recently, two more systems have been developed that can simultaneously replace several nucleotides.

The Technological Basis of smACGmax

The foundation of smACGmax is the CRISPR/Cas9 system, which allows DNA to be cut at specific sites. However, for the purposes of directed evolution, the goal is not to destroy the gene but to alter its sequence in as many ways as possible. To achieve this, the Cas9n protein is used, which makes a cut on only one DNA strand. Inside Cas9n, a complex of several enzymes is embedded: cytosine and adenine deaminases (which break down cytosine and adenine) and a DNA glycosylase that removes damaged bases. To enable guanine removal, a modified version of human glycosylase was added. The resulting system was named smACGmax.

Mechanism of Action

The system works as follows: a gap is created on one DNA strand at the site of the damaged and removed bases, and the second strand, which usually serves as a template for repairing the first, is also cut. In this situation, the base excision repair (BER) system cannot function. Cellular translesion polymerases insert random bases in place of the removed adenine, cytosine, or guanine.

Experimental Application

The system was tested on human cells (HEK293T and HeLa). During the experiment, the HBEGF gene, which encodes a receptor that allows diphtheria toxin to enter the cell, was modified. As a result of the editor's work, numerous random variants of this receptor appeared in the cell cultures. After the toxin was added to the culture medium, only those cells in which the receptor had become insensitive survived.

Method Limitations

Currently, guanine editing works reliably only when a specific nucleotide sequence (the NGR motif) is present nearby, so the efficiency of guanine replacement is lower compared to other bases.