SHIELD · To Shield, or not to Shield: deciphering DNA repair pathway choice after CRISPR-Cas cleavage

To Shield, or not to Shield: deciphering DNA repair pathway choice after CRISPR-Cas cleavage

Human genome editing has recently begun to enter mainstream medicine. However, its widespread application remains limited because of costs, low efficiency and risks of adverse health side effects. Over the past decade, a new technique called CRISPR-Cas has become the leading method for genome editing in laboratories. It makes use of a modified bacterial defense mechanism against foreign DNA. Unlike older technologies, it easily adapts to target different genes—an unthinkable feat in the past. First, it recognizes a specific DNA sequence inside a cell, and then it cleaves the DNA essentially breaking it in two halves creating so-called double-strand DNA break (DSB). The cell then fixes the broken DNA in two ways: it can directly join the two DNA ends together utilizing the Non-Homologous End Joining pathway (NHEJ), or it can use similar DNA as a recipe to repair the break as well as to check for possible missing pieces in the Homology Directed Repair pathway (HDR). If we supply the cell with our own DNA recipe, the cell will then use it to repair the DNA. In this way, we can treat genetic diseases otherwise untreatable by conventional medicine. So why isn’t CRISPR-Cas widely used for gene therapies? Surprisingly, it remains unclear how cells choose which way to repair the DNA after CRISPR-Cas cleavage. Moreover, cells prefer to quickly join the DNA ends together, rather than search for similar DNA, which takes more time. As the cells don’t use a DNA recipe to repair the DNA, we can’t influence the sequence of the repaired DNA. The Shieldin protein complex acts at the DNA breaks and was shown to promote the NHEJ pathway in which the DNA ends are joined directly, not requiring the DNA template (recipe). This project focused on understanding the mechanism behind DNA repair pathway choice after CRISPR-Cas-induced double-strand breaks. By biochemical assays, mass spectrometry and electron microscopy, I investigated the Shieldin complex and its proposed ability to suppress HDR and thus promote NHEJ. Finally, I aimed to assess the Shieldin complex as a potential therapeutic target that would lead to more prevalent DNA break repair by HDR. My results showed that the Shieldin complex preferentially binds to single-stranded DNA, but not to DNA overhangs that are formed at double-stranded DNA breaks. Also, I did not observe the DNA end protecting activity of the Shieldin complex in vitro. With recent findings showing that the Shieldin complex plays a more subtle role at DNA breaks than initially understood (Swift M.L. et al., Nat Struct Mol Biol, 2023), as of now the Shieldin complex does not constitute a good target to enhance the efficiency of the CRISPR-Cas-based genome modifications.

Data: CORDIS, © European Union