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

Abstract

BackgroundThe liverwort Marchantia polymorpha 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 MpEF1α promoter prompted us to establish the TALEN gene targeting technique for Marchantia.ResultsHere, two different TALEN techniques, using custom and self-assembled TALEN constructs, were applied and compared. The MpNOP1 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 Mpnop1-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 MpEF1α promoter.ConclusionConsidering 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.

Conserved redox‐dependent DNA binding of ROXY glutaredoxins with TGA transcription factors

“…Variation in ROXY expression patterns enabled further sub‐ and neofunctionalization processes, which likely contributed to the recruitment of ROXYs into various developmental and stress‐related processes. The availability of molecular tools for M. polymorpha (Ishizaki, Nishihama, Yamato, & Kohchi, ; Kopischke, Schussler, Althoff, & Zachgo, ; Sugano et al., ) enables further functional analyses in one of the most basal land plants. This will help to advance our recently expanding knowledge on redox regulation of plant TF activities (Dietz, ) and can reveal how these processes contributed to adapt plants to novel challenges associated with a terrestrial lifestyle.…”

Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha

“…Recently, a transcription activator-like effector nuclease (TALEN)-based genome editing technology was established in M. polymorpha [24]. We have previously demonstrated that a CRISPR/Cas9-based knockout system, which exploited human-codon-optimized Cas9, can operate in M. polymorpha [25].…”

Deciphering the Evolution and Development of the Cuticle by Studying Lipid Transfer Proteins in Mosses and Liverworts

“…Because the dominant phase of the bryophyte life cycle is the haploid gametophyte, the effects of transgenes can be studied already a few weeks after transformation. There are established protocols for obtaining gene knock-outs or knock-downs via RNAi, Transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, homologous recombination or artificial miRNAs [ 53 , 54 , 55 , 56 , 57 , 58 , 59 ]. There are also many reporter genes and promoters available for gain-of-function, gene expression and protein localization studies [ 60 , 61 , 62 , 63 ].…”