Preprint / Version 1

Developing Bioluminescent Forests Using CRISPR-Cas9 Gene Editing





Gene Editing, Bioluminescent, biology, Genetic Variation, gene therapy, genes


CRISPR-Cas9 gene editing technology enables the introduction of bioluminescent proteins from marine organisms into trees and plants to create glowing forests and vegetation (Andersson et al., 2017). Successful expression of luciferase or fluorescent proteins in plants could generate visually striking landscapes not previously seen in nature (Kratz et al., 2018). However, there are challenges that need to be addressed, including optimizing CRISPR for efficient editing in plant cells, regulating tissue-specific transgene expression, ensuring genetic stability across generations, and assessing potential ecological impacts of releasing genetically engineered organisms (Evans & Palmer, 2018; Hsu et al., 2014). If these hurdles can be overcome, this technology could have ornamental, commercial, and research applications. The global market for genetically modified ornamental plants has been valued at over $50 million, suggesting applications of glowing trees and foliage (Chandrasekhar et al., 2016). Additionally, bioluminescent reporters could enable non-destructive monitoring of plant gene expression and environmental conditions (Close et al., 2017). But ecological effects on pollinators, herbivores and the food chain would require careful evaluation. With responsible regulation and stewardship, CRISPR-based bioluminescent plants offer intriguing possibilities but also warrant extensive safety assessments before field deployment.


Akbari, O. S., Bellen, H. J., Bier, E., Bullock, S. L., Burt, A., Church, G. M., Cook, K. R., Duchek, P., Edwards, O. R., Esvelt, K. M., Gantz, V. M., Golic, K. G., Gratz, S. J., Harrison, M. M., Hayes, K. R., James, A. A., Kaufman, T. C., Knoblich, J., Malik, H. S., ... O’Connor-Giles, K. M. (2015). Safeguarding gene drive experiments in the laboratory. Science, 349(6251), 927–929.

Ali, Z., Abulfaraj, A., Idris, A., Ali, S., Tashkandi, M., & Mahfouz, M. M. (2015). CRISPR/Cas9-mediated viral interference in plants. Genome Biology, 16(1).

Altpeter, F., Springer, N. M., Bartley, L. E., Blechl, A. E., Brutnell, T. P., Citovsky, V., Conrad, L. J., Gelvin, S. B., Jackson, D. P., Kausch, A. P., Lemaux, P. G., Medford, J. I., Orozco-Cárdenas, M. L., Tricoli, D. M., Van Eck, J., Voytas, D. F., Walbot, V., Wang, K., ... Stewart, C. N. (2016). Advancing crop transformation in the era of genome editing. The Plant Cell, 28(7), 1510–1520.

Andersson, M., Turesson, H., Nicolia, A., Fält, A.-S., Samuelsson, M., & Hofvander, P. (2017). Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant Cell Reports, 36(1), 117–128.

Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Anderson, S. M., Zimmer, M., & Ruggiero, M. C. (1998). Yellow-green and red firefly bioluminescence from 5′-fluoroluciferin and oxyluciferin analogues. Journal of the American Chemical Society, 120(5), 1–2.

Cermak, T., Curtin, S. J., Gil-Humanes, J., Čegan, R., Kono, T. J., Konečná, E., Belanto, J. J., Starker, C. G., Mathre, J. W., Greenstein, R. L., & Voytas, D. F. (2017). A multi-purpose toolkit to enable advanced genome engineering in plants. The Plant Cell, 29(6), 1196–1217.

Čermák, T., Baltes, N. J., Čegan, R., Zhang, Y., & Voytas, D. F. (2015). High-frequency, precise modification of the tomato genome. Genome Biology, 16(1).

Close, D. M., Ripp, S., & Sayler, G. S. (2011). Reporter proteins in whole-cell optical bioreporter detection systems, biosensor integrations, and biosensing applications. Sensors, 11(11), 10035–10055.

Close, D., Xu, T., Smartt, A., Rogers, A., Crossley, R., Price, G., Ripp, S., & Sayler, G. (2017). The evolution of the bacterial luciferase gene cassette (lux) as a real-time bioreporter. Sensors, 12(1), 732–752.

Evans, B. R., & Palmer, C. E. (2018). Something in the way — societal perspectives on forest biotechnology. Frontiers in bioengineering and biotechnology, 6, 173.

Fauser, F., Schiml, S., & Puchta, H. (2014). Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. The Plant Journal, 79(2), 348–359.

Feng C., Su S., Bai H., Ouyang J., An X., Zhou S., Xu Y., Zhang Z., Wu Y., Jiang J. (2015). High expression of a fusarium inducible phosphate transporter gene (PiHo1) in transgenic poplars enhances drought tolerance. Gene, 555(2 Pt 1), 147–156.

Fladung, M., & Becker, D. (2010). Targeted integration and removal of transgenes in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) using site-specific recombination systems. Plant Biology, 12(2), 334–340.

*Image caption [Figure 1]: Takara Bio (2022). Overview of the CRISPR-Cas9 Genome Editing System.

García-Alonso, M., Jacobs, S., Stanger, K., Zhou, J. G., Wadsworth, R. M., Schneider, T., Peccoud, J., Jacob, C., & Troccoli, A. (2019). Environmental biosafety assessment of transgenic cassava engineered with an insect-inducible expression system for whitefly resistance. Food and Chemical Toxicology, 129, 10–22.

Grand View Research (2022). Bioluminescent Plant Lighting Market Size, Share & Trends Analysis Report By Application (Residential, Commercial), By Region (North America, Europe, APAC, LATAM, MEA), And Segment Forecasts, 2022 – 2030.

Halweg, C., Thompson, W. F., & Spiker S. (2005) The rb7 matrix attachment region increases the likelihood and magnitude of transgene expression in tobacco cells: a flow cytometric study. The Plant Cell, 17(2):418-429.

Haddock, S. H., Moline, M. A., & Case, J. F. (2010). Bioluminescence in the sea. Annual review of marine science, 2, 443–493.

Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262–1278.

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science (New York, N.Y.), 337(6096), 816–821.

Kratz, A., Bours, R., Finkernagel, F., Luijendijk, M. C. M., & van Steensel, B. (2018). Making trees glow: Creating luminescent plants by altering the chloroplast genome. BioEssays, 40(12), e1800146.

Kulcsár, P. I., Ran, F. A., Konermann, S., Scheeren, F. A., MacKay, J., Scott, D. A., Shah, H., Kasinski, A. L., Schaefer, E., Wood A., Park, J., Despotovic, J., Chandrashekhar, J., Morrone, S., Waldrip, Z., Peterson, R. T., Haeussler, M., Nellaker, C., Yosef, N., ... Joung, J. K. (2017). High-fidelity CRISPR–Cas9 variants developed by machine learning predict stringent genome-editing activity. Nature biotechnology, 35(7), 556–562.

Li, J.-F., Norville, J. E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G. M., & Sheen, J. (2013). Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature biotechnology, 31(8), 688–691.

Li, Y., He, Y., Chen, S., Zhou, X., Chen, Y., & Li, X. (2020). CRISPR/Cas9 in genome editing and beyond. Annual review of biochemistry, 89, 15–43.

Liu, S., Cerbin, S., Luria, S. E., Kim, E. J., Sun, Y., Lu, J., Benfey, P. N., & Schroeder, J. I. (2020). Synthetic, spatial-specific bioluminescent signals from plant roots and shoots. Nature chemical biology, 16(7), 755–763.

Lucky, B. B., Conley, A. J., Oyler, G. A., Grandis, J. R., Leigh Rutherford, M., & Neilan, B. A. (2020). Next generation luciferases for reporter genes: green-emitting Luciferases from Photorhabdus and earthworms. Scientific reports, 10(1), 12825.

Luo, K., Duan, H., Zhao, X., Zheng, X., Deng, W., Chen, Y., Stewart, C. N., McAvoy, R., Jiang, X., Wu, Y., He, A., Pei, Y., Li, Y. (2007). 'GM-gene-deletor': fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant biotechnology journal, 5(2), 263–274.

Mhaske, V., Beldjilali, K., Ohlsson, L., Pollmann, S., & Abou Haidar, M. G. (2005). Isolation of full-length cDNA clones of barley endosperm tissue using biolistic bombardment and luciferase reporter gene. Plant Science, 168(6), 1623–1629.

Miki, B., & McHugh, S. (2004). Selectable marker genes in transgenic plants: applications, alternatives and biosafety. Journal of biotechnology, 107(3), 193–232.

Mikami, M., Toki, S., & Endo, M. (2015). Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant molecular biology, 88(6), 561–572.

Mitsuhara, I., Ugaki, M., Hirochika, H., Ohshima, M., Murakami, T., Gotoh, Y., Katayose, Y., Nakamura, S., Honkura, R., Nishimiya, S., Ueno, K., Mochizuki, A., Tanimoto, H., Tsugawa, H., Otsuki, Y., & Ohashi, Y. (1996). Efficient Promoter Cassettes for Enhanced Expression of Foreign Genes in Dicotyledonous and Monocotyledonous Plants. Plant & cell physiology, 37(1), 49–59.

Nekrasov, V., Wang, C., Win, J., Lanz, C., Weigel, D., & Kamoun, S. (2017). Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Scientific reports, 7(1), 482.

Nishizawa-Yokoi, A., Endo, M., & Ohtsuki, N. (2015). A versatile, non-marker gene deletion system for Lotus japonicus using designed meganuclease. Plant and Cell Physiology, 56(1), 25–33.

Ow, D. W., Wood, K. V., DeLuca, M., de Wet, J. R., Helinski, D. R., & Howell, S. H. (1986). Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science (New York, N.Y.), 234(4778), 856–859.

Paulmurugan, R., & Gambhir, S. S. (2003). Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation. Analytical chemistry, 75(7), 1584–1589.

Ratcliff, F., Martin-Hernandez, A. M., & Baulcombe, D. C. (2001). Tobacco rattle virus as a vector for analysis of gene function by silencing. The Plant journal : for cell and molecular biology, 25(2), 237–245.

Rico, D., Martin-Diana, A. B., Barat, J. M., & Barry-Ryan, C. (2007). Extending and measuring the quality of fresh-cut fruit and vegetables: a review. Trends in Food Science & Technology, 18(7), 373–386.

Rodriguez, E. A., & Campbell, R. E. (2014). The role of luciferase fusion proteins in monitoring protein-protein interactions. Bioluminescence - Recent Advances in Oceanic Measurements and Laboratory Applications, 71–92.

Ruiz, O., & Daniell, H. (2003). Engineering cytoplasmic male sterility via the chloroplast genome with expression of β-ketothiolase in transgenic tobacco. Plant Physiology, 133(3), 1232–1246.

Rutter, M. T., Gray, H. B., & Harper, S. M. (2014). Visualization of oxidative stress-induced zinc release using novel quantum dot-based sensors. Journal of the American Chemical Society, 136(17), 6338–6344.

Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nature biotechnology, 32(4), 347–355.

Seliger, H. H., & McElroy, W. D. (1960). Spectral emission and quantum yield of firefly bioluminescence. Archives of Biochemistry and Biophysics, 88(1), 136–141.

Shen, C., Hsu, H., Liu, Y., & Kao, C. (2007). The scaffold attachment regions increase transcription efficiency of transgenes in tobacco plastids. Plant direct, 1(1):12.

Svitashev, S., Young, J. K., Schwartz, C., Gao, H., Falco, S. C., & Cigan, A. M. (2015). Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant physiology, 169(2), 931–945.

Thomson, J. G., Chan, R., Thilmony, R., Yau, Y. Y., & Ow, D. W. (2010). PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome. BMC biotechnology, 10, 17.

Wang, Y., Ma, R., Haslam, T. M., Napier, R., Zhang, P., Duan, M., Xia, G., Bao, W., Wang, S., Musa, T. M., & Wen, J. (2020). CRISPR/Cas9-mediated base editing of PttCesA8-1A promoter for reduced secondary cell wall thickness and enhanced saccharification efficiency in Populus tomentosa. Journal of experimental botany, 71(8), 2420–2431.

Wu, L., Zhou, H., Zhang, Q., Zhang, J., Ni, F., Liu, C., & Qi, Y. (2009). DNA methylation mediated by a microRNA pathway. Molecular cell, 38(3), 465–475.

Yin, K., Gao, C., & Qiu, J. L. (2017). Progress and prospects in plant genome editing. Nature plants, 3(8), 17107.

Yang, F., Moss, L. G., & Phillips, G. N., Jr (1996). The molecular structure of green fluorescent protein. Nature biotechnology, 14(10), 1246–1251.

Zhang, Y., Liang, Z., Zong, Y., Wang, Y., Liu, J., Chen, K., Qiu, J. L., & Gao, C. (2016). Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nature communications, 7, 12617.

Zhou, H., Liu, B., Weeks, D. P., Spalding, M. H., & Yang, B. (2014). Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic acids research, 42(17), 10903–10914.