Agrobacterium- and a single Cas9-sgRNA transcript system-mediated high efficiency gene editing in perennial ryegrass

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species.

[1]  H. Kim,et al.  Spatiotemporal control of CRISPR/Cas9 gene editing , 2021, Signal Transduction and Targeted Therapy.

[2]  Yunde Zhao,et al.  A reporter for noninvasively monitoring gene expression and plant transformation , 2020, Horticulture research.

[3]  K. Jung,et al.  Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook , 2020, International journal of molecular sciences.

[4]  Y. Ran,et al.  Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system , 2020, Plant biotechnology journal.

[5]  Baohui Liu,et al.  Perspectives on the application of genome editing technologies in crop breeding. , 2019, Molecular plant.

[6]  Tao Zhang,et al.  Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing , 2019, Plant biotechnology journal.

[7]  Tingting Gu,et al.  A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants , 2018, Horticulture Research.

[8]  Yannick Jacob,et al.  Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter , 2017, Plant Cell Reports.

[9]  Yiping Qi,et al.  A Single Transcript CRISPR-Cas9 System for Efficient Genome Editing in Plants. , 2016, Molecular plant.

[10]  K. Shinozaki,et al.  Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants , 2016, Scientific Reports.

[11]  Leif Skøt,et al.  Implementation of Genomic Prediction in Lolium perenne (L.) Breeding Populations , 2016, Front. Plant Sci..

[12]  Q. Xie,et al.  High-Efficiency Genome Editing in Arabidopsis Using YAO Promoter-Driven CRISPR/Cas9 System. , 2015, Molecular plant.

[13]  Jacqueline D. Campbell,et al.  A synteny-based draft genome sequence of the forage grass Lolium perenne. , 2015, The Plant journal : for cell and molecular biology.

[14]  Wei Liu,et al.  A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. , 2015, Molecular plant.

[15]  Xuecheng Wang,et al.  Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation , 2015, Genome Biology.

[16]  Yi Li,et al.  Genome-editing technologies and their potential application in horticultural crop breeding , 2015, Horticulture Research.

[17]  Yuriko Osakabe,et al.  Genome editing with engineered nucleases in plants. , 2015, Plant & cell physiology.

[18]  R. Qu,et al.  Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection , 2013, Plant Cell, Tissue and Organ Culture (PCTOC).

[19]  R. Qu,et al.  Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses , 2013, Plant Molecular Biology.

[20]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[21]  Z.-X. Zhang,et al.  Overexpression of Nicotianamine Synthase (NAS) Gene Results in Enhanced Drought Tolerance in Perennial Ryegrass , 2008 .

[22]  Z.-X. Zhang,et al.  Callus Induction and Plant Regeneration in Eleven Perennial Ryegrass Cultivars , 2006 .

[23]  Arlene A. Wise,et al.  Three methods for the introduction of foreign DNA into Agrobacterium. , 2006, Methods in molecular biology.

[24]  W. A. Harwood,et al.  A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques , 2005, Plant Cell Reports.

[25]  Bingru Huang,et al.  Physiological Responses to Heat Stress Alone or in Combination with Drought: A Comparison between Tall Fescue and Perennial Ryegrass , 2001 .

[26]  M. Wright,et al.  Recent advances in the transformation of plants. , 1999, Trends in plant science.

[27]  R. Emmons.  Turfgrass Science and Management , 1984 .