Periodic Reporting for period 1 - SHIELD (To Shield, or not to Shield: deciphering DNA repair pathway choice after CRISPR-Cas cleavage)
Periodo di rendicontazione: 2022-09-01 al 2024-08-31
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.
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.
I have successfully produced Shieldin complex in both insect Hi5 cells and human derived HEK293T cells. I optimized purification protocol to achieve high purity samples. In addition, I produced the Shieldin complex together with its interaction partner protein Rif1.
AlphaFold structural models of the Shieldin complex hinted at a high degree of conformational flexibility among individual domains and subunits. Mass spectrometry crosslinking revealed that the complex is highly flexible and likely partially unstructured, which was also confirmed by serial negative stain electron microscopy. This property of the Shieldin complex does not seem to change even upon interaction with DNA.
The Shieldin complex prefers to bind single-stranded DNA and 3’overhangs. On the other hand, only modest binding was detected for double-stranded DNA. Shieldin complex was additionally tested for its ability to shield the 3’overhang against resection in exonuclease competition assays in vitro. However, in this experimental setup the Shieldin complex failed to protect the DNA.
Overall, my results show that Shieldin complex prefers to bind to the ssDNA in vitro, but it is not capable of protecting the DNA from resection on its own or while interacting with Rif1.
AlphaFold structural models of the Shieldin complex hinted at a high degree of conformational flexibility among individual domains and subunits. Mass spectrometry crosslinking revealed that the complex is highly flexible and likely partially unstructured, which was also confirmed by serial negative stain electron microscopy. This property of the Shieldin complex does not seem to change even upon interaction with DNA.
The Shieldin complex prefers to bind single-stranded DNA and 3’overhangs. On the other hand, only modest binding was detected for double-stranded DNA. Shieldin complex was additionally tested for its ability to shield the 3’overhang against resection in exonuclease competition assays in vitro. However, in this experimental setup the Shieldin complex failed to protect the DNA.
Overall, my results show that Shieldin complex prefers to bind to the ssDNA in vitro, but it is not capable of protecting the DNA from resection on its own or while interacting with Rif1.
Successful production of the Shieldin complex in native environment in human derived HEK cells allowed me to explore the DNA binding and DNA protecting properties of the complex. The findings showed that Shieldin complex interacts preferably with single-stranded DNA, but not with dsDNA or 3’overhangs found at the sites of DNA double-strand breaks. Also, in exonuclease competition assays, it does not protect the 3’overhangs against further DNA resection in vitro. Including additional Rif1 (1-350) protein that helps recruit the Shieldin complex to the DNA breaks yielded the same results. Altogether, the results contradict the so far accepted view that Shieldin binds to and protects the overhangs from further processing. Analysis by electron microscopy, mass photometry, and mass spectrometry showed that the complex is very flexible and contains large unstructured regions. That makes it harder to identify specific regions that might be targeted by therapeutics. As Shieldin is also involved in antibody maturation, its function might be restricted to a very specific set of conditions. Altogether, Shieldin complex does not seem to be suitable target for increasing the efficiency of the CRISPR-Cas based genome editing techniques.