Glue instead of scissors
A group of researchers from Columbia University has found a way to fix one of the main shortcomings of modern gene editing tools, including CRISPR, and has proposed a new approach to genetic engineering and gene therapy.
The new INTEGRATE technology described in the study uses transposons ("jumping genes") of bacteria to create any DNA sequence without having to cut its chain. Modern gene editing tools are DNA scissors, but cutting the chain and further stitching it often leads to errors. The new INTEGRATE technology is more like a molecular glue: instead of breaking the integrity of the chain and then restoring it, INTEGRATE directly introduces pre-created DNA into the genome.
Existing tools are capricious
The method of editing the cell genome presented in the study is similar to using a text editor to format a huge document, but this software has its own intelligence. Typically, researchers want to make a small change to one particular sequence of nitrogenous bases, leaving the rest of the genome intact. Currently, the CRISPR-Cas tool is used for this, it crosses both DNA chains in a certain area and inserts the required sequence there. For clarity, the process can be compared to adding an entire paragraph to a block of text in a text editor.
The main drawback of the method lies in the process of stitching the chain – the cell does it on its own. Often, cells repair the DNA molecule incorrectly or with errors, some cells cannot provide DNA repair at all. In addition, DNA breakdown causes a reaction to damage, which has undesirable negative effects.
These disadvantages make gene editing difficult or impossible in some cell types and severely limit the ability of researchers to make accurate and safe changes to the genome.
INTEGRATE uses "jumping genes"
This study is designed to solve the problems of the existing DNA editing system and uses weakened vibrio cholera bacteria to dispense with the help of the cell itself.
To find a new tool for gene editing, Sam Sternberg and his graduate students closely studied the transposons, or "jumping genes" found in the cholera vibrio. This transposon was introduced into the classical CRISPR-Cas system.
The researchers found that the transposon integrates into certain sites in the bacterial genome not by cutting DNA into two parts, but by using an integrase, an enzyme used to introduce the transposon into the genome.
This discovery was used to create a genome editing tool that can be programmed to insert any DNA sequence into any part of the bacterial genome. Like CRISPR, integrase finds the corresponding segment using a guide RNA.
By reprogramming the guide RNA, scientists were able to precisely control the localization of donor DNA integration. By replacing the transposon base sequence with the required one, they were able to introduce DNA segments up to 10,000 bases long into the bacterial genome. Thus, the INTEGRATE technology, unlike other integrase-based editing tools, is the first fully programmable DNA insertion system studied to date.
Sequencing of the edited bacterial genome confirmed that useful DNA segments were inserted accurately, without additional copies on non-target sites.
Updated gene editing
In the INTEGRATE system, a set of enzymes can carry out the entire process of DNA insertion, precisely introducing the necessary sequence of bases into a given section of the cell genome, without the need for the cell itself to participate in the restoration of the chain.
INTEGRATE technology provides the same degree of programmability and ease of use as CRISPR-Cas9, but without the side effects associated with DNA breaks.
Next steps
Sternberg's group plans to test INTEGRATE not only in bacterial cells, but also in many others, including mammalian cells.
The authors argue that there are good reasons to expect the same success of the INTEGRATE system in mammalian cells. This will open the door for basic research and possible clinical applications.
Article by S. E. Klompe et al. Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration is published in the journal Nature.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on the materials of Columbia University Irving Medical Center: New Gene Editor Harnesses Jumping Genes for Precise DNA Integration.