CRISPR/Cas9-Mediated Knockout of Physcomitrella patens Phytochromes

Area of Science:

• Plant molecular biology
• Genetics and genomics
• Moss biology

Background:

• Physcomitrella offers a model system for plant gene function studies.
• Understanding gene redundancy is crucial for deciphering complex phenotypes.
• CRISPR/Cas9 technology provides precise genome editing tools.

Purpose of the Study:

• To establish and present robust CRISPR/Cas9-based gene disruption and excision protocols for Physcomitrella.
• To enable simultaneous targeting of multiple gene loci for functional genomics.
• To facilitate the investigation of phytochrome (PHY) gene functions in mosses.

Main Methods:

• CRISPR/Cas9 system utilizing single guide RNA (sgRNA) and Cas9 nuclease to induce double-strand breaks (DSBs).
• Non-homologous end joining (NHEJ) for gene disruption via insertions/deletions (indels).
• Homologous recombination-assisted repair for precise gene or fragment excision.
• CRISPOR online tool for target site prediction and sgRNA design.
• Gateway cloning system for plasmid construction.
• Moss cotransformation with Cas9 and selectable marker plasmids.
• PCR fingerprinting for transformant analysis.

Main Results:

• Successful gene disruption and excision in Physcomitrella demonstrated.
• Protocol adaptable for multiplex gene targeting.
• Methodology validated through targeting of phytochrome (PHY) gene loci.
• Efficient generation and identification of gene-edited moss lines.

Conclusions:

• CRISPR/Cas9 provides a versatile platform for genetic manipulation in Physcomitrella.
• The developed protocols are valuable for dissecting gene function, especially in cases of functional redundancy.
• This work advances the study of phytochrome biology and potentially other complex traits in mosses and higher plants.