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

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.

Cas12a (formerly known as Cpf1) is a bacterial enzyme that cleaves foreign DNA as part of an adaptive immune system comprising Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) and CRISPR-associated (cas) genes. In complex with an ~44 nt CRISPR RNA (crRNA), Cas12a binds by base-pairing between the crRNA and a DNA protospacer sequence, resulting in DNA unwinding and formation of a DNA-RNA heteroduplex (R-loop). R-loop formation activates dsDNA cleavage by a RuvC domain. The relative simplicity of the Type V CRISPR Cas12a effectors and their crRNA means that they have great potential as gene editing enzymes, and there is evidence that Cas12a has lower off-target cleavage activity in cells compared to Cas9. Multiple Cas12a structures have been determined and some rudimentary enzymatic characterisation undertaken, and yet the fundamental mechanistic properties of R-loop formation and nuclease activation remain unclear. We aim to address this by combining biochemical, biophysical and single-molecule kinetic assays. We will first measure the kinetics and thermodynamics of R-loop formation using a Magnetic Tweezers microscope. We will study the influence of the protospacer adjacent motif (PAM), crRNA, and protospacer sequences, to determine how R-loop stability and size are controlled, and how these properties are affected by DNA-RNA mismatches or changes in nucleoprotein contacts. FRET between labels on Cas12a and the DNA will be used to follow domain motions that link stable R-loop formation with RuvC activation. We will explore how RuvC engages with the DNA strands sequentially to produce ordered dsDNA cleavage. Finally, we will use high-throughput nanopore sequencing to map single DNA cleavage events, to determine how changes in the R-loop structure alter cleavage site position. The knowledge we obtain will be vital to the future development of Cas12a tools for genome surgery, particularly in predicting the effects of mismatches.

The use of CRISPR enzymes for gene editing, transcriptional regulation or modulating epigenetic patterning has increased rapidly, and they have become commonly-used lab tools. This project seeks to determine exactly how Cas12a operates, so that the community can more accurately predict how the enzyme will deal with a wide range of different genomic targets.

Academic researchers across disparate fields, from synthetic to cell biology, will therefore benefit. In the short term, the information we obtain will be valuable to researchers interested in CRISPR biology and its influence on prokaryotic evolutionary dynamics, where CRISPR may regulate horizontal gene transfer and the spread of antibiotic resistance. Longer term, our work will influence researchers who are striving to improve Cas12a or to develop new genome surgery tools. Understanding the basic enzymatic mechanism will be a necessary part of re-engineering efforts, particularly knowledge about how mismatches influence activity. There are huge potential societal benefits of safe, targeted genome therapy. Therefore, social scientists can also benefit indirectly from this research, by discussion with our team about the ethical implications of gene editing. This in turn can help in the development of policy, which will be an area of growing importance to the gene editing field.

Biotechnology firms who are interested in developing gene therapy or utilising CRISPR tools will benefit from this project as it seeks to understand how Cas12a nucleases recognise sequences in a specific manner. The increased knowledge base will allow such companies to improve and develop their products, for example by supporting decisions to switch from Type II to Type V enzymes. Some companies are aiming to improve CRISPR by nucleotide modification and the smaller crRNA of Cas12a has a benefit of being amenable to phosphoramidite synthesis. As well as conventional pharmaceutical companies that are now routinely using CRISPR, there are also companies dedicated to CRISPR development and/or gene editing, such as Editas Medicine or Caribou Bioscience Inc. Our research can directly influence their applied use of Cas12a.

In the longer term, healthcare professionals will benefit from the research as it will add to the growing knowledge of the operation of CRISPR enzymes; gene editing approaches are already being applied in clinical settings and a principal goal of this project is to provide greater mechanistic understanding to reduce off target cleavage in a predictable manner.

The PDRA will gain valuable skills in the application of state-of-the-art biochemical, biophysical and single-molecule methods. The development of analytical tools and kinetic models will also boost their mathematical knowledge. The experience they gain will give them a technical edge in their future careers and will add to the UK infrastructure in workers capable of applying interdisciplinary techniques. The PDRA will also take part in training undergraduates, giving them experience in quantitative analysis that they can apply in a wide-range of other occupations.

Finally, the general public and patient groups will benefit from the proposed research. The design of safer enzymes for genome editing will contribute to economic and health quality of life of those living in the UK. We will therefore strive to increase the public's understanding of the science surrounding this project and of the potential of gene editing in general. We will do this through workshops, through University Open Days and Post-offer Days, and by participation in outreach (e.g. Pint of Science). These interactions will be facilitated by the discussions with social scientist about the ethics of gene editing. In turn this can provide a basis to help formulate policy (similar to deliberations about mitochondrial replacement therapy - so-called Three Parent Families).