Periodic Reporting for period 3 - ARGO (The Quest of the Argonautes - from Myth to Reality)
Okres sprawozdawczy: 2022-07-01 do 2023-12-31
Fundamental research & Impact for Society
The genomics era has resulted in an enormous release of uncharacterized protein-encoding genes. The on-going optimization of comparative genomics tools has resulted in a steadily expanding set of predictions. Since 2006, my research team initiated a research line on a predicted novel prokaryotic defense system called CRISPR-Cas. Over the years we have actively participated in the molecular characterization of several CRISPR-Cas types: Class 1, Type I (Cas3/Cascade) and Type III (Cas10/Cmr and Cas10/Csm), Class 2, Type II (ThermoCas9) and Type V (Cas12a). Recently, we have initiated the characterization and engineering of ThermoCas9 and of novel miniature CRISPR-Cas12 variants. In addition, since 2010, we have studied prokaryotic Argonaute (pAgo) variants, distantly related to the well-characterized eukaryotic Argonaute. This has revealed spectacular mechanistic variations. In the ARGO project we aim to further explore these distantly related Argonautes and Cas nucleases. After a thorough biochemical characterization of selected guided nucleases, both Argonautes and CRISPR-associated nucleases, we aim at engineering the functionality of selected nucleases through an integrated rational & random approach, i.e. by designed tinkering of domains, and by a novel laboratory evolution approach. Eventually, based on their specific features, natural & synthetic nucleases will be selected for the development of innovative genome editing applications. Genome editing can be instrumental for a wide range of applications, from microbial biotechnology, crop improvement to human gene therapy and diagnostics of pathogens and diseases.
Overall objectives
Using an approach that combines molecular biology and biochemistry with microbiology, ARGO is an integrated project: (i) characterization the mechanistic variations of Ago-variants (Med13 & pAgo), (ii) design enzyme with unprecedented functionalities, (iii) develop an original laboratory evolution approach & (iv) establish innovative applications. Specifically, five complementary objectives are being executed: one on elucidating the control mechanism by a eukaryotic Ago-variant, and four on the exploration, engineering and exploitation of prokaryotic Ago-variants:
• (Aim-1) Dissect role & mode of action of an Ago-like regulator
• (Aim-2) Reveal function & mechanism of natural pAgo/Cas variants
• (Aim-3) Vary Ago/Cas functionality by Rational Design
• (Aim-4) Improve Ago/Cas performance by Laboratory Evolution
• (Aim-5) Develop Ago/Cas-based applications
The genomics era has resulted in an enormous release of uncharacterized protein-encoding genes. The on-going optimization of comparative genomics tools has resulted in a steadily expanding set of predictions. Since 2006, my research team initiated a research line on a predicted novel prokaryotic defense system called CRISPR-Cas. Over the years we have actively participated in the molecular characterization of several CRISPR-Cas types: Class 1, Type I (Cas3/Cascade) and Type III (Cas10/Cmr and Cas10/Csm), Class 2, Type II (ThermoCas9) and Type V (Cas12a). Recently, we have initiated the characterization and engineering of ThermoCas9 and of novel miniature CRISPR-Cas12 variants. In addition, since 2010, we have studied prokaryotic Argonaute (pAgo) variants, distantly related to the well-characterized eukaryotic Argonaute. This has revealed spectacular mechanistic variations. In the ARGO project we aim to further explore these distantly related Argonautes and Cas nucleases. After a thorough biochemical characterization of selected guided nucleases, both Argonautes and CRISPR-associated nucleases, we aim at engineering the functionality of selected nucleases through an integrated rational & random approach, i.e. by designed tinkering of domains, and by a novel laboratory evolution approach. Eventually, based on their specific features, natural & synthetic nucleases will be selected for the development of innovative genome editing applications. Genome editing can be instrumental for a wide range of applications, from microbial biotechnology, crop improvement to human gene therapy and diagnostics of pathogens and diseases.
Overall objectives
Using an approach that combines molecular biology and biochemistry with microbiology, ARGO is an integrated project: (i) characterization the mechanistic variations of Ago-variants (Med13 & pAgo), (ii) design enzyme with unprecedented functionalities, (iii) develop an original laboratory evolution approach & (iv) establish innovative applications. Specifically, five complementary objectives are being executed: one on elucidating the control mechanism by a eukaryotic Ago-variant, and four on the exploration, engineering and exploitation of prokaryotic Ago-variants:
• (Aim-1) Dissect role & mode of action of an Ago-like regulator
• (Aim-2) Reveal function & mechanism of natural pAgo/Cas variants
• (Aim-3) Vary Ago/Cas functionality by Rational Design
• (Aim-4) Improve Ago/Cas performance by Laboratory Evolution
• (Aim-5) Develop Ago/Cas-based applications
The ARGO project has had a very successful in the first and second period (1-7-2019 / 31-12-2021), as can be judged from the output in terms of publications in respectable scientific journals, as well as several patents 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 been widened a bit from just Argonaute (-like) proteins to RNA/DNA-guided nucleases that share the same catalytic domain (PIWI/RuvC). This implies that apart from the proposed Argonautes (like CbAgo and Mediator/Med13), also selected CRISPR-associated nucleases are studied in the course of the project. With the recent arrival of Postdoc Dr Isabelle Zink, and the anticipated arrival of Dr Laureen Mertens (Sept 2022), the Argonaute sub-projects will get more attention.
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.