TALEN-mediated genome-editing approaches in the liverwort yield high efficiencies for targeted mutagenesis.

Background: The liverwort occupies a crucial position in land plant evolution and provides the opportunity to investigate adaptations to a terrestrial plant life style. Marchantia reverse genetic analyses have thus far been conducted by employing a homologous recombination approach, which yields an efficiency of around 3%. Availability of the characterized and suitable endogenous Mp promoter prompted us to establish the TALEN gene targeting technique for Marchantia.

Results: Here, two different TALEN techniques, using custom and self-assembled TALEN constructs, were applied and compared. The Mp gene was selected as a candidate gene, as the respective knockout mutant has been shown to lack air chamber formation, representing an easily traceable phenotype. We demonstrate that both TALEN approaches are successful in Marchantia yielding high gene targeting efficiencies of over 20%. Investigation of selected G1 up to G4 generations proved the stability of the knockout mutants. In 392 analyzed T1 plants, no additional phenotypes were observed and only one chimeric knockout plant was detected after an extended cultivation period. Interestingly, two out of the 24 sequenced mutants harbored indels causing in-frame mutations and revealed novel Mp-related phenotypes. This demonstrates the potential to detect crucial amino acids and motives of targeted proteins, which is of special interest for essential genes where full knockouts are lethal. The FastTALE™ TALEN assembly kit enables the rapid assembly and ligation of the TALEN arms within half a day. For transformations, custom and assembled constructs were subcloned into Marchantia binary vectors possessing the Mp promoter.

Conclusion: Considering time, costs and practicability, the assembly TALEN approach represents a rapid and highly efficient gene targeting system to generate Marchantia knockout mutants, which can be further adapted for future advanced genome-editing applications.