CRISPR-Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella) patens.

Area of Science:

• Molecular Biology
• Plant Science
• Genome Editing

Background:

• Precise genome editing was previously limited to a few organisms.
• CRISPR-Cas9 technology has significantly expanded molecular toolkits across many organisms and cell types.
• Prior to CRISPR-Cas9, P. patens relied on homologous recombination for DNA integration, which required selection and limited editing types.

Purpose of the Study:

• To describe a streamlined protocol for generating diverse genome edits in P. patens using CRISPR-Cas9.
• To detail methods for Cas9/sgRNA expression construct generation, homology template design, transformation, and genotyping.
• To expand molecular manipulation capabilities in P. patens.

Main Methods:

• Generation of Cas9/sgRNA expression constructs (including shortcut methods).
• Design of homology-directed repair (HDR) templates (oligonucleotide-based and plasmid-based).
• Transformation of CRISPR vectors into P. patens protoplasts and subsequent identification of edited plants.

Main Results:

• A comprehensive protocol for CRISPR-Cas9 mediated genome editing in P. patens is presented.
• The protocol facilitates streamlined generation of various genome edits.
• Efficient transformation and genotyping methods are described for rapid identification of edited plants.

Conclusions:

• CRISPR-Cas9 technology significantly enhances genome editing capabilities in P. patens.
• This protocol provides a robust and efficient method for diverse genetic modifications in P. patens.
• The described methods accelerate research and applications involving genome editing in this plant model.