Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

Key message Biolistic delivery into pollen. Abstract In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.

Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize

Recent advances in the development of CRISPR-Cas genome editing technologies have made it possible to perform targeted mutagenesis and precise gene replacement in crop plants. CRISPR-Cas9 and CRISPR-Cas12a are two main types of widely used genome editing systems. However, when CRISPR-Cas12a editing machinery is expressed from a transgene, some chromosomal targets encountered low editing frequency in important crops like maize and soybean. Here, we report efficient methods to directly generate genome edited lines by delivering Cas12a-gRNA ribonucleoprotein complex (RNP) to immature maize embryos through particle bombardment in an elite maize variety. Genome edited lines were obtained at ~7% frequency without any selection during regeneration via biolistic delivery of Cas12a RNP into immature embryos. Strikingly, the gene editing rate was increased to 60% on average and up to 100% in some experiments when the Cas12a RNP was co-delivered with a PMI selectable marker gene cassette and the induced callus cultures were selected with mannose. We also show that use of higher activity Cas12a mutants resulted in improved editing efficiency in more recalcitrant target sequence. The advances described here provide useful tools for genetic improvement of maize.

An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue

Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT-2020. In addition, the same approach is applied to test the efficacy of multiple gRNAs for CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

In recent years, genome-editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding.Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need forAgrobacterium-mediated transfection and tissue culture, by using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, theArabidopsis UBQ10promoter was found to be the most suitable for driving expression of the delivered gene in pollen tubes. We also evaluated delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9 introduced pollen tubes but were not detected in the negative control. Bombarded pollen germinated pollen tubes on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.

An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue

Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT®-2020. In addition, the same approach is applied to test the efficacy of CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.

High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice

An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.