Arabidopsis glutamate:glyoxylate aminotransferase 1 (Ler) mutants generated by CRISPR/Cas9 and their characteristics
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) technology provides an efficient tool for editing the genomes of plants, animals and microorganisms. Glutamate:glyoxylate aminotransferase 1 (GGAT1) is a key enzyme in the photorespiration pathway; however, its regulation mechanism is largely unknown. Given that EMS-mutagenized ggat1 (Col-0 background) M2 pools have been generated, ggat1 (Ler background) should be very useful in the positional cloning of suppressor and/or enhancer genes of GGAT1. Unfortunately, such ggat1 (Ler) mutants are not currently available. In this study, CRISPR/Cas9 was used to generate ggat1 (Ler) mutants. Two GGAT1 target single-guide RNAs (sgRNAs) were constructed into pYLCRISPR/Cas9P35S-N, and flowering Arabidopsis (Ler) plants were transformed using an Agrobacterium tumefaciens-mediated floral dip protocol. Eleven chimeric and two heterozygous GGAT1-edited T1 lines of target 1 were separately screened from positive transgenic lines. Two ggat1 homozygous mutants, CTC-deletion and T-deletion at target 1, were generated from T2 generations of the 13 T1 lines. The edited mutation sites were found to be stable through generations regardless of whether the T-DNA was present. In addition, the genetic segregation of the mutation sites obeyed the Mendelian single gene segregation rule, and no mutations were detected at the possible off-target site. Also, the two independent ggat1 mutants had similar photorespiration phenotypes and down-regulated GGAT enzyme activity. Together, these results indicate that genetically stable ggat1 (Ler) mutants were generated by CRISPR/Cas9 genome editing, and these mutants will be used to promote the positional cloning of suppressor and/or enhancer genes of GGAT1 in our subsequent study.
KeywordsGenome editing technology CRISPR/Cas9 GGAT1 Photorespiration Arabidopsis
Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9
Glutamate:glyoxylate aminotransferase 1
Protospacer adjacent motif
Ribulose bisphosphate carboxylase/oxygenase
The Arabidopsis Information Resource
Transcription activator-like effector nucleases
Zinc finger nucleases
This research was sponsored by the Science and Technology Funding of Guangzhou (Grant Nos. 201607020006 and 201707010032).
YL and XZ performed the experiments. XP and XH designed the experiments and performed the bioinformatics analysis. XH prepared the manuscript. All authors read and approved the final version of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
All the materials and data reported in this article are available upon request.
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