Periodic Reporting for period 3 - MIGHTY_RNA (Repurposing small RNA from ciliates for genome editing: single-molecule study)
Período documentado: 2022-05-01 hasta 2023-10-31
Genome editing is an essential tool for life sciences and biotechnology. The big wave of CRISPR changed the paradigm of life sciences by opening up an effective way of genetic engineering. Cas9-genome editing is easy to implement because nearly any DNA sequence can be targeted by altering the sequence of small guide RNA of Cas9. However, it has limitations in biotechnology and medical applications. Numerous studies showed that CRISPR suffers from off-target effects. Its use for genome editing is also restricted due to its sequence requirement for targeting, and its poor accessibility to structured genome. Recent studies also suggested that it might trigger immune responses in human cells. Here we have aimed to explore new genome editing systems that have a potential to overcome the aforementioned limitations. We examine how small-regulatory RNA systems can accurately recognize target nucleic acids. This fundamental study will help generating a new genome editing system. In the short term, its impact will reach fundamental research and, in the long run as far as medical sciences.
We have studied several potential genome editing systems. We have done biochemistry and single-molecule experiments on new Argonaute systems discovered from prokaryotes, new PIWI systems from eukaryotes and archaea, and new prokaryotic CRISPR systems. We have explored the biophysics of these new systems and looked for the potential of using them for genome editing. The outcome of the studies have been published in prominent journals (Cui et al, Nature Communications, 2019; Filius et al, Nano Letters, 2020; Ruijtenberg et al, Nature Structural and Molecular Biology, 2020; Bastiaanssen et al, RNA Biology, 2021). The results have been presented in more than 10 seminars, workshops, and conferences nationally and internationally. It includes an invited talk in the Keystone Symposium.
This project involves the development of a new single-molecule technique--integration of single-molecule fluorescence with DNA deep sequencing. This exciting technique development is on track. When fully accomplished, it will open up the genome-wide and transcriptome-wide single-molecule studies for the first time. The use of the tool will be actively disseminated via publishing protocols in peer reviewed journals, sharing the software via public databases, and training external users.
This project involves the development of a new single-molecule technique--integration of single-molecule fluorescence with DNA deep sequencing. This exciting technique development is on track. When fully accomplished, it will open up the genome-wide and transcriptome-wide single-molecule studies for the first time. The use of the tool will be actively disseminated via publishing protocols in peer reviewed journals, sharing the software via public databases, and training external users.
All aforementioned benefits of new genome editing systems will make impact to scientists in life sciences and medical doctors for clinical trials. The impact of our research will reach the whole society such as patients with genetic disease. Our research will set an example of how fundamental research can be used for long-term applications relevant to the society. In addition, the development of the cutting-edge high throughput single-molecule fluorescence method will become a revolutionary tool in single-molecule biophysics.