A mechanistic framework for DNA recognition and cleavage by Type V CRISPR-Cas effector nucleases

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It was demonstrated over 30 years ago that new genetic information could be inserted into the genomes of cultured human cells in the lab. This revolutionary discovery opened up the possibility of not only studying gene function but also correcting genetic mistakes that lead to disease. However, the process was inefficient, requiring millions of cells to be screened to find just one that had swapped a gene. It was realised that this process could be improved by introducing an enzyme into the cells that could break the DNA at the gene of interest (i.e. to cut both DNA strands using a so-called nuclease), and allowing the cell's natural DNA repair processes to do the rest. However, this required the development of "molecular scissors" that would cut just one gene amongst billions of other potential targets. Most research efforts concentrated on protein re-engineering of enzymes to recognise a user-defined sequence of DNA bases (and thus a unique gene). However, these tools were difficult to work with and the search continued for simpler programmable enzymes.A breakthrough came with the discovery in the early 2000s of bacterial enzyme systems that prevent viral infection, called Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) and CRISPR-associated (cas) genes. CRISPR-Cas systems had molecular scissors that recognised DNA in a unique way; a CRISPR nuclease separated the DNA strands and inserted an RNA molecule (called a "crRNA") to read out the DNA sequence, so producing a DNA-RNA hybrid (called an "R-loop"). CRISPR systems were easier to reprogram as only the RNA had to be changed and this was trivial for scientists compared to enzyme re-engineering. The CRISPR-led revolution in gene editing ignited in 2012 with the characterisation of CRISPR Cas9 and the first demonstrations of gene editing by Cas9 in human cell culture. A great deal of research has now been done using Cas9: it has been adapted for a wide range of genetic engineering functions, both in the lab and in the clinic; and we have a good understanding of how it works. Cas9 is fast becoming a common tool for basic, synthetic and clinical research.The Cas12a family of CRISPR nucleases have a similar biological function to Cas9 but were first characterised only in 2015. The structures of Cas12a enzymes are similar to Cas9 and they also appear to recognise gene sequences using crRNA-guided R-loops. However, there are key differences in the protein structures and we currently do not understand exactly how Cas12a works. It is important that we do so as it appears that Cas12a may be a better gene editing tool than Cas9. It has lower off-target cleavage in cells, meaning that the molecular scissors cut in the wrong place less often. Why this is the case is not known. The overall goal of this project is to establish more clearly how Cas12a forms an R-loop and cleaves the DNA, and how this is influenced by the crRNA and DNA sequences.To study Cas12a, we will use a combination of biochemistry and biophysics using purified proteins, DNA and RNA. Our principal technique is Magnetic Tweezers Microscopy. This "single-molecule" approach can observe R-loop formation by just one enzyme on one DNA molecule. We will seek to understand how the R-loop forms, how this is influenced by Cas12a, how changes in the crRNA affect the dynamics, and how incorrect pairing between the DNA and crRNA alter how the scissors cut the DNA. We will follow the DNA cleavage process and map where the cleavage occurs using a single-molecule DNA sequencing technique, called nanopore sequencing. And we will follow how different parts of the Cas12a protein move in relation to one another and to the DNA, by labelling with fluorescent markers. The culmination of these studies will be a fuller understanding of how Cas12a works and why it is more accurate. This will form the basis of future studies to improve Cas12a, and to further adapt it as the next generation of gene editing tools.