A CRISPR/LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens.

Area of Science:

• Molecular Biology
• Plant Science
• Genetics

Background:

• Programmable nucleases like CRISPR/Cas12a offer efficient, specific, and flexible genome editing across organisms.
• CRISPR/Cas12a allows multiplex gene editing by assembling multiple CRISPR RNAs (crRNAs) in a single vector.
• The efficacy of CRISPR/Cas12a in the non-vascular plant Physcomitrella patens remains largely uncharacterized.

Purpose of the Study:

• To investigate the activity and efficiency of CRISPR/Cas12a for genome editing in Physcomitrella patens.
• To demonstrate the capability of simultaneous targeting of multiple loci using CRISPR/Cas12a in P. patens.
• To expand the available genome editing toolkit for P. patens research.

Main Methods:

• Co-delivery of LbCas12a and a crRNA expression cassette in vivo into P. patens.
• Utilizing CRISPR/LbCas12a to target multiple genetic loci simultaneously.
• Analysis of mutation frequencies and types induced by CRISPR/LbCas12a.

Main Results:

• LbCas12a demonstrated high efficiency in targeting multiple loci simultaneously in P. patens.
• Mutation frequencies at single loci ranged from 26.5% to 100%.
• Diverse deletions were identified as the predominant mutation type.

Conclusions:

• CRISPR/Cas12a is an effective genome editing tool for P. patens.
• The developed method facilitates the creation of multiplex loss-of-function mutants in P. patens.
• This research expands genome editing capabilities for studying gene function in non-vascular plants.