Periodic Reporting for period 1 - VISCREEN (Engineering viral vectors for high-throughput CRISPR genetic screens in plants)
Período documentado: 2023-04-01 hasta 2025-03-31
CRISPR-Cas genome editing offers new opportunities to carry out genetic screens and understand fundamental concepts in plant genetics. The simultaneous delivery of thousands of guide RNAs (gRNAs) into cells allows to rapidly create an ordered mutant collection overcoming functional redundancy in plant genomes (mainly due to polyploidization and genome duplications). While standard CRISPR screens rely on plasmid pools delivered via conventional transformation techniques, the delivery of CRISPR-Cas components using plant viral vectors offers a promising alternative for a rapid and efficient modification without genetic transformation. Combining virus-induced genome editing (VIGE) with CRISPR represents a novel approach to generate large mutant populations in non-transformable cultivars.
The VISCREEN project aims to develop and expand the current toolbox of viral vectors so that VIGE can be implemented as a powerful, easy-to-use tool for plant genome engineering. I will approach two specific objectives: (1) develop a high-throughput genome editing system using Barley stripe mosaic virus (BSMV) for wheat and maize; and (2) engineer a set of viral vectors for gene editing in soybean.
The VISCREEN project aims to develop and expand the current toolbox of viral vectors so that VIGE can be implemented as a powerful, easy-to-use tool for plant genome engineering. I will approach two specific objectives: (1) develop a high-throughput genome editing system using Barley stripe mosaic virus (BSMV) for wheat and maize; and (2) engineer a set of viral vectors for gene editing in soybean.
Objective 1: Develop a high-throughput genome editing system in wheat and maize. Although the objective was not fully achieved, I performed these tasks:
• Adaptation of BSMV subgenomes into a suitable binary plasmid suitable for in planta expression
• Homology-based selection of target genes in wheat and maize, followed by sgRNA design
• In silico design of BSMV-sgRNA construct for multiplex editing and molecular cloning
• Genotyping of transgenic Cas9 wheat lines (T1 and T2 generations), selection of homozygous lines and seed harvest
• Inoculation of wheat and maize plants with recombinant BSMV vectors
Objective 2: Engineer and characterize a set of viral vectors for legume functional genomics. Although the objective was not fully achieved, I performed these tasks:
• Selection of target genes with breeding potential in soybean, followed by shRNA design
• In silico design of recombinant vectors for the mobile Cas9-sgRNA system and molecular cloning
• Adaptation of BSMV subgenomes into a suitable binary plasmid suitable for in planta expression
• Homology-based selection of target genes in wheat and maize, followed by sgRNA design
• In silico design of BSMV-sgRNA construct for multiplex editing and molecular cloning
• Genotyping of transgenic Cas9 wheat lines (T1 and T2 generations), selection of homozygous lines and seed harvest
• Inoculation of wheat and maize plants with recombinant BSMV vectors
Objective 2: Engineer and characterize a set of viral vectors for legume functional genomics. Although the objective was not fully achieved, I performed these tasks:
• Selection of target genes with breeding potential in soybean, followed by shRNA design
• In silico design of recombinant vectors for the mobile Cas9-sgRNA system and molecular cloning
No innovative results were derived from the project itself. However, a fruitful inter-institutional collaboration with the IBMCP (Valencia, Spain) led to the development of a viral vector based on potato virus X (PVX) for genome editing in tomato. This approach offers new opportunities for studying genetic diversity in tomato and for precision breeding of horticultural traits. Moreover, it highlights the potential of VIGE for transitioning from proof-of-concept applications in model plants to the fast-track improvement of Solanaceae crop production systems, contributing to enhanced global and local human nutrition and food security.
Additional research and efforts are required to demonstrate that VIGE technologies meet the legal and biosafety requirements to be commercially exploited as an easy-to-use tool for crop improvement in agriculture.
Additional research and efforts are required to demonstrate that VIGE technologies meet the legal and biosafety requirements to be commercially exploited as an easy-to-use tool for crop improvement in agriculture.