Precise templated editing using endogenous DNA polymerases
The paper introduces THOR, a CRISPR-Cas9-based technology that utilizes DNA-tailed guides to recruit endogenous DNA polymerases for precise, exogenous-polymerase-free genome editing across diverse targets and cell models.
Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer
The human genome is often described as a vast library of instructions, written in a chemical code that dictates how our bodies grow and function. Sometimes, a single letter in this code is wrong, leading to disease. For years, scientists have been developing tools to find and fix these errors, a field known as genome editing. The most famous of these tools acts like a pair of molecular scissors, cutting the DNA at a specific location. Once the cut is made, the cell's natural repair machinery kicks in to stitch the strands back together. However, this natural repair process is often messy, leading to random deletions or insertions that can cause more harm than good. To achieve precise corrections, scientists previously had to bring in their own specialized enzymes to copy a correct template directly onto the DNA. This added a layer of complexity, making the tools larger and harder to deliver into cells. The central question for researchers has been whether the cell's own internal machinery, which it uses every day to copy and repair its genetic material, could be coaxed into performing this precise editing task without the need for extra, external enzymes.
A team of researchers at AstraZeneca and collaborating institutions has developed a new approach called THOR, which stands for Template Homology-Oriented Repair, to answer this question. Instead of relying on a foreign enzyme to copy a correction, this method uses a modified guide molecule that carries a short piece of DNA attached to its end. When the guide finds its target in the genome, it creates a small nick or cut. The attached DNA piece then acts as a primer, inviting the cell's own natural DNA polymerases—enzymes that the cell already possesses and uses for normal maintenance—to bind and copy the desired edit from the guide onto the genome. The researchers demonstrated that this system works effectively in human cells, successfully installing specific genetic changes at multiple locations without needing to supply an external copying enzyme. This finding suggests that the cell's internal repair tools are sufficient to perform high-precision editing if they are given the right starting point.
The team tested this concept by comparing their new THOR system against existing methods that rely on reverse transcriptase, an enzyme that copies RNA into DNA. In their experiments, they used human cells grown in a laboratory and targeted several different genes, including those associated with sickle cell disease. They found that the THOR system, which uses the cell's own DNA polymerases, could install precise edits with a level of accuracy comparable to the best existing tools. Crucially, because the system does not require a complex external enzyme, the guide molecule is simpler to manufacture and package. The researchers also observed that this method avoided a specific type of unwanted byproduct seen in other systems, where parts of the guide molecule itself get accidentally copied into the genome. By using a DNA tail on the guide, the system ensures that the cell's natural enzymes stop copying once they reach the end of the instruction, preventing these errors.
Further investigation revealed how the system behaves under different conditions and in different types of cells. The researchers tested the method in blood stem cells, which are critical for treating blood disorders, and found that the technique worked well, especially when the cells were stimulated to grow before the editing took place. They also explored whether adding chemical inhibitors could improve the quality of the edits by blocking the cell's messy repair pathways. They discovered that blocking certain repair enzymes increased the proportion of precise edits, making the outcome cleaner. However, they also found that the system did not suffer from the same limitations as other tools regarding the availability of building blocks inside the cell, suggesting it is robust across different cellular environments. The team also tested delivering the system using lipid nanoparticles, tiny fat bubbles used to carry genetic material into cells, and confirmed that the method remains functional even when not directly injected into the cell, though the efficiency was slightly lower than direct injection.
While the new method shows great promise, the researchers noted that it is not a perfect replacement for all existing tools in every situation. In some specific locations within the genome, the system was less efficient than other methods, and the performance varied depending on the type of edit being made. The study suggests that the success of the method depends heavily on the specific design of the guide molecule and the interaction between the guide and the cell's natural enzymes. The researchers also pointed out that the stability of the guide molecule could be improved to make the system even more effective. Overall, the work establishes a new pathway for precise genome editing that leverages the cell's own biology rather than fighting against it. By proving that endogenous DNA polymerases can be directed to perform precise templated editing, the study opens the door to simpler, more versatile tools for correcting genetic errors, potentially making these therapies easier to develop and deliver in the future.
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