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Longevity Briefs: Fixing Large DNA Mutations With New CRISPR Tools

Posted on 20 October 2021

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Longevity briefs provides a short summary of novel research in biology, medicine, or biotechnology that caught the attention of our researchers in Oxford, due to its potential to improve our health, wellbeing, and longevity.

Why is this research important: CRISPR/Cas9 is a powerful gene editing tool that has only improved since its development in 2012. Recent advancements such as prime editing in 2019 mean that CRISPR can now be used to edit the DNA sequence with greater precision than ever before, giving scientists the ability to delete, add, or swap out single nucleotides – the individual building blocks that make up the genetic code. However, even these newer CRISPR technologies are limited in the size of the changes they can make at once, struggling when it comes to cutting out anything longer than around 100 nucleotides (for reference, human genes are commonly around 27 000 nucleotides long). This makes genetic mutations involving the large changes (such as the duplication or rearrangement of a long sequence of the genetic code) out of bounds for treatment with CRISPR.

How traditional CRISPR/Cas9 gene editing works
Johan Jarnestad
The Royal Swedish Academy of Sciences

What did the researchers do: Two teams of researchers working independently have developed CRISPR technologies that can delete sections of DNA up to around 10 000 nucleotides in length. The first team at the University of Massachusetts named their method PEDAR (for PE-Cas9-based deletion and repair). They did this by adding an additional guide RNA, the molecule that acts as a navigator for the other components of the editing system. These two guide RNAs allowed the researchers to pick two places at which the Cas9 enzyme (the ‘scissors’ component) would cut, resulting in the removal of everything in between those two points. The final component, reverse transcriptase, was then able to bridge the gap by extending new ‘sticky’ single DNA strands from each cutting site, using the guide RNA as a template for what to build. Once those ‘sticky’ strands meet, the deletion and repair is completed.

The second team at the University of Washington used a similar approach involving two guide RNAs to cut the DNA strand in two locations. The main difference was that while the Massachusetts team used a Cas9 that cuts both strands of the double helix, the Washington team used a Cas9 that only cuts one – this is the same kind of Cas9 used in prime editing. This leaves two ‘sticky’ strands of DNA temporarily intact to bridge the gap between the cut sites.

Key takeaway(s) from this research: We now have two new CRISPR technologies that can make large DNA edits. These technologies have not been extensively tested, and it will be a while before they have any practical application in human diseases, though early testing in mice appears promising. The Massachusetts team was able to correct tyrosinemia-causing mutations in mice, which involves deleting about 1300 nucleotides (tyrosinemia is a rare genetic condition that leads to liver damage and cancer). This technology may also help scientists study the function of ‘junk’ DNA, by allowing them to delete portions of these very large regions and observe how gene expression is affected.

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      Deletion and replacement of long genomic sequences using prime editing:

      Precise genomic deletions using paired prime editing:

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