Generation of stable nulliplex autopolyploid lines of Arabidopsis thaliana using CRISPR/Cas9 genome editing
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RNA-guided endonuclease-mediated targeted mutagenesis using the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system has been successful at targeting specific loci for modification in plants. While polyploidy is an evolutionary mechanism enabling plant adaptation, the analysis of gene function in polyploid plants has been limited due to challenges associated with generating polyploid knockout mutants for all gene copies in polyploid plant lines. This study investigated whether CRISPR/Cas9 mediated targeted mutagenesis can generate nulliplex tetraploid mutant lines in Arabidopsis thaliana, while also comparing the relative efficiency of targeted mutagenesis in tetraploid (4x) versus diploid (2x) backgrounds. Using CRISPR/Cas9 genome editing to generate knockout alleles of the TTG1 gene, we demonstrate that homozygous nulliplex mutants can be directly generated in tetraploid Arabidopsis thaliana plants. CRISPR/Cas9 genome editing now provides a route to more efficient generation of polyploid mutants for improving understanding of genome dosage effects in plants.
KeywordsGenome editing CRISPR/Cas9 Arabidopsis thaliana Polyploidy Genome dosage
We are grateful to Prof. Chen, Qi-Jun for kindly providing the vector pHEE401 for subsequent vector construction in this study. This work was supported by grant funding from Science Foundation Ireland (SFI) to CS (Principal Investigator Grant 13/IA/1820), and a postdoctoral fellowship from the Irish Research Council (IRC) to MMH.
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Conflict of interest
The authors declare that they have no conflict of interest.
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