Periodic Reporting for period 4 - ARGO (The Quest of the Argonautes - from Myth to Reality)
Reporting period: 2024-01-01 to 2024-06-30
Fundamental research & Impact for Society
A series of spectacular scientific discoveries and technological advances in the second half of the 20th century have provided the basis for the ongoing genome editing revolution. The elucidation of structural and functional features of DNA and RNA was followed by pioneering studies on genome editing, that altogether allowed for the development of the Molecular Biotechnology field. Since then, several decades followed during which progress of scientific insights and technological methods continued at an overwhelming pace. Fundamental insights into microbial host-virus interactions led to the development of tools for genome editing, initially using restriction enzymes and more recently the revolutionary Argonaute and CRISPR-Cas technology. In this project, novel fundamental features have been revealed, and based on that some new applications have been developed.
Fundamental research (often without clear ideas on how that may benefit society) is crucial for gaining understanding the interplay of chemical and physical features that ultimately determine the functionality of complex biological systems. The purpose of such research is not always obvious, and in many cases merely driven by scientific curiosity. Still, there are many examples of fundamental research that generates huge societal impact. Beautiful examples are the basic insights on two defence/control system Argonaute and CRISPR-Cas, that has resulted in the extremely fast development of different genome editing applications, that are being used in both diagnostics (sensitive detection of pathogens and disease related mutations) and genome editing (from crop optimization to human gene therapy).
The ARGO project has focussed on discovering new Argonaute and CRISPR-Cas related nucleases, characterizing them to reveal molecular mechanistic features, and last but not least to try to develop new applications. As indicated below, ample dissemination efforts have been realized, including lectures, publications and a patent application. Overall, it is concluded that the project has been very successful !
A series of spectacular scientific discoveries and technological advances in the second half of the 20th century have provided the basis for the ongoing genome editing revolution. The elucidation of structural and functional features of DNA and RNA was followed by pioneering studies on genome editing, that altogether allowed for the development of the Molecular Biotechnology field. Since then, several decades followed during which progress of scientific insights and technological methods continued at an overwhelming pace. Fundamental insights into microbial host-virus interactions led to the development of tools for genome editing, initially using restriction enzymes and more recently the revolutionary Argonaute and CRISPR-Cas technology. In this project, novel fundamental features have been revealed, and based on that some new applications have been developed.
Fundamental research (often without clear ideas on how that may benefit society) is crucial for gaining understanding the interplay of chemical and physical features that ultimately determine the functionality of complex biological systems. The purpose of such research is not always obvious, and in many cases merely driven by scientific curiosity. Still, there are many examples of fundamental research that generates huge societal impact. Beautiful examples are the basic insights on two defence/control system Argonaute and CRISPR-Cas, that has resulted in the extremely fast development of different genome editing applications, that are being used in both diagnostics (sensitive detection of pathogens and disease related mutations) and genome editing (from crop optimization to human gene therapy).
The ARGO project has focussed on discovering new Argonaute and CRISPR-Cas related nucleases, characterizing them to reveal molecular mechanistic features, and last but not least to try to develop new applications. As indicated below, ample dissemination efforts have been realized, including lectures, publications and a patent application. Overall, it is concluded that the project has been very successful !
The ARGO project is considered very successful from June 2019-June 2024, as can be judged from the output in terms of publications in respectable scientific journals, as well a patent application, all with the involvement of PhD students/Postdocs/PIs. As stated in the original proposal, the technologies in general, and the to-be-developed methods for obtaining variant enzymes as well as the screening/selection thereof, is applicable for Argonautes and the related CRISPR-associated nucleases. Compared to the original proposal, the scope has indeed been widened a bit from just Argonaute (-like) proteins to RNA/DNA-guided nucleases (both CRISPR-Cas nucleases, and their transposon-associated ancestor (TnpB) that share the same catalytic domain (PIWI/RuvC). This implies that apart from the proposed Argonautes (like CbAgo, TtAgo, PfAgo), also selected CRISPR-associated nucleases are studied in the course of the project (ThermoCas9, Cas12m, Cas12f, TnpB).
Overview of Results, Exploitation & Dissimination
The ARGO project has led to a series of publications in high impact, open access Journals, as well as to a single patent application (describing a new methos for detecting disease-related nucleic acid variations in crude human samples. As indicated in more detail below (Table 1, Table 2) the work has been presented and discussed at several international scientific meetings, both physical and on-line (during the COVID pandemic), as well as several occasions organized to reach out to the general public in the Netherlands (varying from articles in local newspapers to the Paradiso theatre, and from tv interviews to a music festival (Lowlands)).
Table 1. Involvement of recruited scientists (PD, PhD) in different sub-Projects of ARGO project.
Output as indicated (PUB, publication(s); PAT, patent; LEC, lectures); PUB*=PUB in preparation.
# Name Ago Cas9 Cas12 Type III Output
1 Ioannis Mougiakos (PD) + PUB, LEC
2 Jorrit Hegge (PD) + PUB, LEC
3 Prarthana Mohanraju (PD) + + PUB, LEC
4 Maartje Luteijn (PD) + PUB
5 Wen Wu (PD) + + PUB, LEC
6 Isabelle Zink (PD) + PUB*, LEC, PAT
7 Laureen Mertens (PD) + PUB*
8 Thomas Swartjes (PD) + PUB
9 Jurre Steens (PhD) + + PUB, LEC
10 Eugenios Bouzetos (PhD) + + PUB
Table 2. Involvement of staff scientists (PI, principle investigator; AP, Associate Prof, TC, Technician)
in different sub-Projects of ARGO project. Output as indicated (PUB, publication(s);
PAT, patent; LEC, lectures); PUB*=PUB in preparation.
# Name Ago Cas9 Cas12 Type III Output
1. John van der Oost (PI) + + + + PUB, PAT, LEC
2. Raymond Staals (AP) + + + PUB, PAT, LEC
3. Rob Joosten (TC) + PUB
Overview of Results, Exploitation & Dissimination
The ARGO project has led to a series of publications in high impact, open access Journals, as well as to a single patent application (describing a new methos for detecting disease-related nucleic acid variations in crude human samples. As indicated in more detail below (Table 1, Table 2) the work has been presented and discussed at several international scientific meetings, both physical and on-line (during the COVID pandemic), as well as several occasions organized to reach out to the general public in the Netherlands (varying from articles in local newspapers to the Paradiso theatre, and from tv interviews to a music festival (Lowlands)).
Table 1. Involvement of recruited scientists (PD, PhD) in different sub-Projects of ARGO project.
Output as indicated (PUB, publication(s); PAT, patent; LEC, lectures); PUB*=PUB in preparation.
# Name Ago Cas9 Cas12 Type III Output
1 Ioannis Mougiakos (PD) + PUB, LEC
2 Jorrit Hegge (PD) + PUB, LEC
3 Prarthana Mohanraju (PD) + + PUB, LEC
4 Maartje Luteijn (PD) + PUB
5 Wen Wu (PD) + + PUB, LEC
6 Isabelle Zink (PD) + PUB*, LEC, PAT
7 Laureen Mertens (PD) + PUB*
8 Thomas Swartjes (PD) + PUB
9 Jurre Steens (PhD) + + PUB, LEC
10 Eugenios Bouzetos (PhD) + + PUB
Table 2. Involvement of staff scientists (PI, principle investigator; AP, Associate Prof, TC, Technician)
in different sub-Projects of ARGO project. Output as indicated (PUB, publication(s);
PAT, patent; LEC, lectures); PUB*=PUB in preparation.
# Name Ago Cas9 Cas12 Type III Output
1. John van der Oost (PI) + + + + PUB, PAT, LEC
2. Raymond Staals (AP) + + + PUB, PAT, LEC
3. Rob Joosten (TC) + PUB
Progress in the different sub-projects includes in-depth analysis of biological function of selected Argonaute and Cas enzymes (Aim-2), and of the biochemical features of natural and synthetic enzymes that are the result of rational design and engineering (Aims 2/3). In addition, steps towards applications have been made (Aim 3). Last but not least, insights gained in in vitro, microfluidics-assisted screening/selection systems (Aims 4/5), that will be instrumental for the proposed functional improvements through laboratory evolution. Overall, it is concluded that substantial progress has been made.