CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the...

Area of Science:

• Plant Biotechnology
• Genome Engineering
• Molecular Biology

Background:

• CRISPR-Cas9 technology enables targeted genome modification using single-guide RNAs (sgRNAs).
• Its application has been limited in nonvascular plants, hindering genetic studies in this group.

Purpose of the Study:

• To report the first successful application of CRISPR-Cas9 genome engineering in the moss Physcomitrella patens.
• To investigate DNA repair mechanisms following CRISPR-Cas9-induced double-strand breaks in this species.

Main Methods:

• Designed sgRNAs targeting the endogenous PpAPT reporter gene in Physcomitrella patens.
• Transformed moss protoplasts with Cas9 and sgRNAs, assessing gene inactivation via 2-fluoroadenine resistance.
• Analyzed DNA repair outcomes, including alternative end-joining (alt-EJ) and homology-driven repair (HDR), with and without donor DNA and PpRAD51.

Main Results:

• CRISPR-Cas9 mediated mutagenesis of the PpAPT gene in approximately 2% of regenerated moss plants, primarily through deletions via alt-EJ.
• In the presence of donor DNA, transgene integration occurred via HDR, with Cas9-induced breaks also repaired by illegitimate recombination.
• Approximately 30% of HDR events occurred independently of the PpRAD51 protein, suggesting alternative HDR pathways.

Conclusions:

• CRISPR-Cas9 is an effective tool for genome engineering in the nonvascular plant Physcomitrella patens.
• Cas9-induced DNA breaks are repaired through multiple pathways, including alt-EJ, HDR, and illegitimate recombination.
• CRISPR-Cas9-mediated HDR in moss is partially independent of the canonical RAD51-dependent homologous recombination pathway.