Direct protein delivery into intact Arabidopsis cells for genome engineering - PubMed (original) (raw)
Direct protein delivery into intact Arabidopsis cells for genome engineering
Yuichi Furuhata et al. Sci Rep. 2024.
Abstract
Intracellular delivery of biomolecules is a prerequisite for genetic techniques such as recombinant engineering and genome editing. Realizing the full potential of this technology requires the development of safe and effective methods for delivering protein tools into cells. In this study, we demonstrated the spontaneous internalization of exogenous proteins into intact cells and root tissue of whole plants of Arabidopsis thaliana. We termed this internalization phenomenon as protein Delivery Independent of Vehicles or Equipment (DIVE), which efficiently delivered genome engineering proteins including Cre recombinase and zinc-finger nucleases (ZFN) into plant cells. Using protein DIVE, we achieved less toxic protein delivery than electroporation with up to 94% efficiency in Arabidopsis cell culture and 19% genome modification in Arabidopsis plants that was maintained in regenerated tissue. This work illustrates the potential of protein DIVE for a wide range of applications, including genome engineering in plants.
© 2024. The Author(s).
Conflict of interest statement
The authors declare no competing interests.
Figures
Fig. 1
Cre protein delivery into T87 cells. (a) Schematic diagram of Cre reporter design before and after Cre-mediated recombination constructed as described in our previous study. The construct T87-xGxGUS includes an expression cassette encoding the gene for green fluorescence protein (GFP) followed by a transcription termination signal and the gene for ß-glucuronidase (GUS) into the genome of T87 cell line. After Cre-mediated recombination between the two loxP sites flanking the GFP gene, T87-xGxGUS cells, originally expressing GFP, expressed GUS and, thus, appeared blue in the presence of X-Glucuronide substrate. GFP, pA, and GUS indicate green fluorescent protein, terminator polyadenylation signal, and ß-glucuronidase, respectively. (b) GUS staining of T87-xGxGUS cells treated with 1 μM Cre protein using Opti-MEM I and NT1HF medium. GUS staining was performed 2 days after the treatment. Control indicates T87-xGxGUS cells without the DIVE treatment including the buffer and the Cre protein. Scale bar represents 200 μm.
Fig. 2
Effect of incubation temperature on DIVE efficiency. (a) GUS staining of T87-xGxGUS cells incubated with 1 μM Cre protein at different temperatures. The cell number was normalized based on their volume. The cells were incubated with 1 μM Cre protein in Opti-MEM I or NT1HF at the indicated temperatures for 1 h. GUS staining was performed 2 days after Cre treatment. Scale bar represents 200 μm. (b) The total GUS activity of the cells in panel a. GUS activity was determined by the fluorescence level of catalyzed 6-chloro-4-methylumbelliferyl β-D-glucuronide 2 days after Cre treatment. P values were obtained using Student’s t-test. (c) Viability of the cells in panel a. CCK8 assay was performed 2 days after incubation. CCK8 activity, i,e., absorbance at 450 nm, was indicated. Control indicates T87-xGxGUS cells without the DIVE treatment including the buffer and the Cre protein. Values shown are the mean ± SE of n = 3.
Fig. 3
Quantification of protein DIVE efficiency. (a) GUS staining of protoplasts generated from T87-xGxGUS cells with Cre treatment. Cells were incubated with 5 μM Cre protein in Opti-MEM I or NT1HF for 1 h. Protoplasts were constructed and stained 2 days after Cre treatment. Scale bar represents 40 μm. (b) Quantification of the percentage of GUS-positive cells in panel a. Values shown are the mean ± SE of n = 3. In total, 416, 402, and 450 cells were used for the quantification of control, Opti-MEM I, and NT1HF samples, respectively. P values versus control were determined by Tukey’s test (n = 3 independent experiments each). (c) Agarose gel electrophoresis of genomic PCR products from T87-xGxGUS cells. The 1307-bp and 151-bp fragments represent the reporter gene cassette before and after Cre-mediated recombination, respectively. A half arrow indicates a primer binding site. Control indicates T87-xGxGUS cells without the DIVE treatment including the buffer and the Cre protein. Values shown are mean ± SE of the ratio of bands after recombination, n = 3. A full-length gel image is presented in Supplementary Fig. 18.
Fig. 4
ZFN protein delivery into T87 cells. (a) Schematic diagram of ZFN reporter design before and after ZFN-mediated SSA in T87-SSA500-ZFN(C5R) cell line. GUS, GU, US, and pA indicate ß-glucuronidase, N-terminal fragment of GUS, C-terminal fragment of GUS, and terminator polyadenylation signal, respectively. (b) GUS staining of T87-SSA500-ZFN(C5R) cells treated with 0.5, 1.0, and 2.0 μM ZFN protein using Opti-MEM I and NT1HF medium. GUS staining was performed 2 days after the treatment. Control indicates T87-SSA500-ZFN(C5R) cells without the DIVE treatment including the buffer and the ZFN protein. Scale bar represents 200 μm.
Fig. 5
Cre protein DIVE in A. thaliana plants. (a) GUS staining of A. thaliana Col-0 xGxGUS plants treated with 5 μM Cre protein using Opti-MEM I and NT1HF medium. GUS staining was performed 2 weeks after Cre treatment. Control indicates untreated plants. Scale bar represents 500 μm. (b) Close-up view of panel a. Scale bar represents 200 μm.
Fig. 6
Calli induction from A. thaliana root tissue after Cre-mediated recombination. (a) Schematic diagram of calli induction from A. thaliana Col-0 xGxGUS roots with Cre treatment. (b) Agarose gel electrophoresis of genomic PCR products from calli generated from A. thaliana Col-0 xGxGUS plants with Cre treatment. The 1307-bp and 151-bp fragments represent the reporter gene cassette before and after Cre-mediated recombination, respectively. M indicates DNA ladder marker. A half arrow indicates a primer binding site.
Fig. 7
Luminescence quantification of GeLgBiT-HiBiT complementation in vitro and in vivo. (a) Schematic diagram of GeLgBiT-HiBiT complementation assay. LgBiT and HiBiT are the large and small fragments of NanoLuc luciferase. Only when these two fragments are directly associated, an active luciferase is reconstituted resulting in oxidation of furimazine and luminescence. In our SLC assay, mNeonGreen is fused to LgBiT (GeLgBiT) to shift the emission wavelength of bioluminescence suitable for plant cells. This allows monitoring of the internalization kinetics of HBNCre(wt), a Cre protein fused to HiBiT, in GeLgBiT-expressing cells and plants with high sensitivity. (b, c) Luminescence kinetics of (b) T87-GeLgBiT and (c) T87-NLS-GeLgBiT cells with HiBiT-tagged Cre treatment. Cells were incubated with Nano-Glo Live Cell Substrate in NT1HF for 5 min. After incubation, HBNCre(wt) (final concentration of 0.5–2 µM) in NT1HF was added and luminescence was measured. (d) Luminescence image of A. thaliana Col-0 NLS-GeLgBiT plants after the addition of Nano-Glo Live Cell Substrate and HBNCre(wt) protein. A. thaliana Col-0 NLS-GeLgBiT plants were soaked in a mixture of the indicated concentration of HBNCre(wt) protein and Nano-Glo Live Cell Substrate solution. Luminescence imaging was performed immediately after soaking.
References
- Clough, S. J. & Bent, A. F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J.16, 735–743 (1998). - PubMed
- Opabode, J. T. Agrobacterium-mediated transformation of plants: Emerging factors that influence efficiency. BMBR1, 12–20 (2006).
- Woo, J. W. et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat. Biotechnol.33, 1162–1164 (2015). - PubMed
MeSH terms
Substances
LinkOut - more resources
Full Text Sources