Plant gene editing
Plant Gene Editing
Introduction
Genome editing is one of the most rapidly advancing technologies in this century. It opens many possibilities that were previously deemed unachievable. Gene editing is the ability to manipulate the genome of an organism by inserting, deleting, or editing existing DNA in the organism’s genome. Plant gene editing is key to meeting the demand for food, pharmaceuticals, and sustainable energy. Genetic enhancements to plants may improve crop yield, resistance to pests, pathogens, and even herbicides. Currently, there are three main gene editing methods in plants: biolistics, agrobacterium, and using protoplasts. Initially, gene-editing technologies weren’t as effective as they are now and would often lead to off-target mutations in the genome that were unintended. Now, with the invention of CRISPR Cas proteins and a push to move towards DNA-free editing, precise and accurate gene-edits can be made with less adverse reactions.
Current Plant-Gene Editing Methods
Biolistics –
Biolistics or biolistic bombardment is the most versatile method of plant-gene editing currently. By propelling heavy metal particles with DNA or protein bound to it at high speeds using a high-pressure gene gun, the particles can penetrate the cell wall of the plant tissue and release the DNA or protein into the nucleus. Biolistics can be used to transfect any tissue, even mammalian tissues. It also is capable of delivering various payloads such as DNA, protein, mRNA, and even viruses, which is unique to this method. In fact, DNA-free genome editing has already been demonstrated in rice.[1], maize [2] and wheat[3]. However, without using any methods of selecting transformed plants by avoiding DNA-delivery, gene-editing efficiencies are typically lower than 10%. The gene-editing efficiencies can be improved significantly by bombarding protein (ribonucleoprotein) along with a plasmid encoding a selectable marker to help enrich for transformed cells during the selection process. Unfortunately, the use of any DNA leads to high-frequency random DNA insertions in the host’s genome, hence DNA-free editing is preferable[4]. The main disadvantage of this method is the possible damage to the biological tissue during the bombardment, which can be optimized during the experiment for minimal damage.
Agrobacterium Mediated Transformation -
Agrobacterium is the most commonly used method of gene-editing in plants to date and was first reported in the early 1980s[5]. It has been widely adopted into commercial practices and produces most of the GMO food you see at the store. Agrobacterium is unique because it utilizes a hijacking micro-organism to deliver T-DNA directly into the host tissue and subsequently, the genome. This method is controlled and often leads to minimal DNA copy numbers per cell, which is preferred for stable transformations of the biological tissue into transformed plantlets. “The Agrobacterium system is attractive because of the ease of the protocol coupled with minimal equipment costs. Moreover, transgenic plants obtained by this method often contain simple copy insertions[6].” More recently, agrobacterium has been utilized to deliver CRISPR Cas9 plasmids while effectively evaluating the performance of several gRNAs reliably. Researchers used agrobacterium in rice to target multiple genes (OsDERF1, OsEPSPS, OsMYB5, OsPMS3, OsPDS OsROC5, OsYSA and OsSPP, OsMYB1) successfully[7][8][9]. The main downside of this method is the limitation of the delivery vesicle, only allowing for DNA to be delivered which always leads in off-target editing effects in the genome and therefore regulation issues.
Protoplasts (PEG-Mediated Transfection, Electroporation, & Lipofection)-
Another method utilized to edit the genomes of plants involves directly removing the cell wall from the plant cell, these are called protoplasts. The main difficulty in gene-editing plants is the rigid cell wall which must be circumvented, however once the cell wall is removed, alternative gene-editing methods like electroporation or lipofection can be used to deliver genes to the protoplasts more efficiently. This method offers a quick analysis of editing efficiencies, only 1-3 days after protoplast transfection. Often, this method is utilized to quickly evaluate the editing efficiencies of different gRNAs, prior to using a model system. To date, regeneration methods from protoplasts to transformed plantlets has been established in lettuce[10][11], potato,[12] tobacco,[13] carrot,[14] petunia,[15] cabbage,[16] maize[17] and rice[18]. The downside to using protoplasts universally is the fact that method is limited to very select plant species, the regeneration procedure is challenging, and the efficiency is still low[19]. More recently, PEG-mediated protoplast transfection was used to deliver cas9 ribonucleoproteins (RNPs) into rice zygotes revealing an editing efficiency of 14-64% and demonstrating a new pathway of DNA-free delivery[20].
Biological Gene-Editing Reagents
Although DNA is the most commonly used editing reagent, there are few reagents that can be used to edit genes. DNA, protein, and mRNA can all be utilized to edit DNA. DNA has frequently been used as a gene-editing tool to edit DNA however, off-target effects have been noticed due to the long half-life of DNA in the cell, overexpressing Cas9 proteins in the genome. More recently, researchers have been trying to utilize protein and mRNA to edit genes, due to their transient nature in the cell. Protein has been the most promising alternative, whereas mRNA has been found difficult to handle due to its low stability[21]
Gene-Editing History
Gene editing techniques were first developed in the 1970s however they did not gain much traction until the 1990s due to the extremely low editing efficiencies and other drawbacks of the technology. Most notably, DNA has been often reported to randomly insert into the host genome, causing unwanted mutations to the host.[1]. In 1985, Zinc Finger Nuclease (ZFN) were discovered and was the first technique with high targeted efficiency due to the ability to recognize DNA and bind to a set of three base pairs to create double stranded breaks (DSBs)[22]. This technique was revolutionary at the time, and it wasn’t until 2011 when the next generation of gene-editing technology was invented, TALENs (Kim et al). Transcription activator-like effector nucleases (TALENs) were unique because of their ease of design and the ability to bind to a single nucleotide, making it much more efficient than ZFNs with less off-target effects[18]. However, TALENs are quite a bit larger and can be hard to deliver to different hosts. Regardless, in 2011, Zinc Finger Nuclease (ZFNs), transcription activator-like effector nucleases (TALENs), and mega nucleases were deemed the 2011 Method of the Year by Nature Methods[23]. Remarkably, in 2012 Jennifer Doudna and her team discovered the ability of CRISPR technology which represented the first technology able to provide precise targeted DNA edits[22] The CRISPR-Cas9 system can be engineered to edit eukaryotic DNA by designing guide RNA complementary to the target sequence. Since CRISPRs invention, the first GMO salmon has been sold in Canada in 2015, a human embryo was successfully edited in China in 2015, and the first human clinical trials were a success in 2020[22]. In 2020, Jennifer Doudna and Emmanuelle Charpentier won the Nobel Prize in Chemistry for their findings. Most recently in 2019, Andrew Anzalone discovered a new technology called prime editing[24]. This is the first technique that can perform targeted, small gene insertions in a precise and efficient way without generating double stranded breaks.
How Gene-editing works:
ZFNs, TALENs, and CRISPR all use double stranded breaks (DSBs) to edit and repair DNA. Currently, there are two major pathways cells use to repair double stranded breaks. Non-homologous end joining (NHEJ) and homology directed repair (HDR) are both commonly used, however HDR is preferred.[18]. HDR is extremely precise in DNA repairing due to the ability to use a homologous sequence template to repair the missing sequences in the genome whereas NHEJ is less accurate and doesn’t use a template. More recently discovered, prime editing introduces the ability to precisely edit DNA without inducing DSBs for the first time[24]
References
- ↑ 1.0 1.1 Banakar, Raviraj (2020). "Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene". Rice. Springer. 13 (1): 4. doi:10.1186/s12284-019-0365-z. PMC 6973557 Check
|pmc=value (help). PMID 31965382. - ↑ Svitashev, Sergei (2016). "Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes". Nature Communications. Nature. 7: 13274. Bibcode:2016NatCo...713274S. doi:10.1038/ncomms13274. PMC 5116081. PMID 27848933.
- ↑ Liang, Zhen (2017). "Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes". Nature Communications. Nature. 8: 14261. Bibcode:2017NatCo...814261L. doi:10.1038/ncomms14261. PMC 5253684. PMID 28098143.
- ↑ Banakar, Raviraj (2020). "Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene". Rice. Springer. 13 (1): 4. doi:10.1186/s12284-019-0365-z. PMC 6973557 Check
|pmc=value (help). PMID 31965382. Unknown parameter|s2cid=ignored (help) - ↑ Bevan, M (1984). "Binary Agrobacterium vectors for plant transformation". Nucleic Acids Research. 12 (22): 8711–8721. doi:10.1093/nar/12.22.8711. PMC 320409. PMID 6095209.
- ↑ Hansen, G (1999). "Recent advances in the transformation of plants". Trends in Plant Science. 4 (6): 226–231. doi:10.1016/s1360-1385(99)01412-0. PMID 10366879. Archived from the original on September 21, 2022. Retrieved September 19, 2022. Unknown parameter
|url-status=ignored (help) - ↑ Feng, Zhengyan (2013). "Efficient genome editing in plants using a CRISPR/Cas system". Cell Research. Nature. 23 (10): 1229–1232. doi:10.1038/cr.2013.114. PMC 3790235. PMID 23958582.
- ↑ Zhang, Hui (2014). "The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation". Plant Biotechnology Journal. 12 (6): 797–807. doi:10.1111/pbi.12200. PMID 24854982. Archived from the original on September 20, 2022. Retrieved September 19, 2022. Unknown parameter
|url-status=ignored (help) - ↑ Ghogare, Rishikesh (2021). "Genome editing reagent delivery in plants". Transgenic Research. Springer. 30 (4): 321–335. doi:10.1007/s11248-021-00239-w. PMID 33728594 Check
|pmid=value (help). Archived from the original on September 21, 2022. Retrieved September 19, 2022. Unknown parameter|url-status=ignored (help); Unknown parameter|s2cid=ignored (help) - ↑ Woo, Je (2015). "DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins". Nature Biotechnology. Nature. 33 (11): 1162–1164. doi:10.1038/nbt.3389. PMID 26479191. Archived from the original on September 21, 2022. Retrieved September 19, 2022. Unknown parameter
|url-status=ignored (help); Unknown parameter|s2cid=ignored (help) - ↑ Andersson, Mariette (2018). "Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery". Physiologia Plantarum. PubMed. 164 (4): 378–384. doi:10.1111/ppl.12731. PMID 29572864. Archived from the original on September 20, 2022. Retrieved September 19, 2022. Unknown parameter
|url-status=ignored (help) - ↑ Lin, Choun-sea (2018). "Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration". Plant Biotechnology Journal. PubMed. 16 (7): 1295–1310. doi:10.1111/pbi.12870. PMC 5999315. PMID 29230929.
- ↑ Grzebelus, E. "An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot". Cabi. Plant Cell Tissue and Organ Culture. Archived from the original on October 15, 2022. Retrieved September 19, 2022. Unknown parameter
|url-status=ignored (help) - ↑ Yu, Jihyeon (2021). "Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins". Plant Cell Reports. PubMed. 40 (6): 1037–1045. doi:10.1007/s00299-020-02593-1. PMID 32959126 Check
|pmid=value (help). Archived from the original on September 20, 2022. Retrieved September 19, 2022. Unknown parameter|url-status=ignored (help); Unknown parameter|s2cid=ignored (help) - ↑ Park, Sung (2019). "DNA-free mutagenesis of GIGANTEA in Brassica oleracea var. capitata using CRISPR/Cas9 ribonucleoprotein complexes". Plant Biotechnology Reports. 13 (5): 483–489. doi:10.1007/s11816-019-00585-6. Archived from the original on September 20, 2022. Retrieved September 19, 2022. Unknown parameter
|s2cid=ignored (help); Unknown parameter|url-status=ignored (help) - ↑ Rhodes, C (1988). "Plant Regeneration from Protoplasts Isolated from Embryogenic Maize Cell Cultures". Nature Biotechnology. Nature. 6: 56–60. doi:10.1038/nbt0188-56. Archived from the original on September 25, 2022. Retrieved September 19, 2022. Unknown parameter
|s2cid=ignored (help); Unknown parameter|url-status=ignored (help) - ↑ He, Guangyuan (2006). "An improved system to establish highly embryogenic haploid cell and protoplast cultures from pollen calluses of maize (Zea mays L.)". Plant Cell, Tissue and Organ Culture. Springer. 86: 15–25. doi:10.1007/s11240-006-9091-5. Archived from the original on September 20, 2022. Retrieved September 19, 2022. Unknown parameter
|s2cid=ignored (help); Unknown parameter|url-status=ignored (help) - ↑ 18.0 18.1 18.2 Li, Z (1990). "Efficient plant regeneration from rice protoplasts in general medium". Plant Cell Reports. PubMed. 9 (4): 216–220. doi:10.1007/BF00232183. PMID 24226706. Archived from the original on September 21, 2022. Retrieved September 19, 2022. Unknown parameter
|url-status=ignored (help); Unknown parameter|s2cid=ignored (help) - ↑ Altpeter, Fredy (2016). "Advancing Crop Transformation in the Era of Genome Editing". The Plant Cell. 28 (7): 1510–1520. doi:10.1105/tpc.16.00196. PMC 4981132. PMID 27335450.
- ↑ Toda, Erika (2019). "An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice". Nature Plants. Nature. 5 (4): 363–368. doi:10.1038/s41477-019-0386-z. PMID 30911123. Archived from the original on September 24, 2022. Retrieved September 19, 2022. Unknown parameter
|url-status=ignored (help); Unknown parameter|s2cid=ignored (help) - ↑ Liang, Zhen (2018). "Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins". Nature Protocols. Nature. 13 (3): 413–430. doi:10.1038/nprot.2017.145. PMID 29388938. Archived from the original on September 22, 2022. Retrieved September 29, 2022. Unknown parameter
|url-status=ignored (help); Unknown parameter|s2cid=ignored (help) - ↑ 22.0 22.1 22.2 "History of Genetic Engineering and the Rise of Genome Editing Tools". Synthego. Synthegi. Archived from the original on September 20, 2022. Retrieved September 19, 2022. Unknown parameter
|url-status=ignored (help) - ↑ "Method of the Year 2011". Nature Methods. Nature. 9 (1): 1. 2012. doi:10.1038/nmeth.1852. PMID 22312634. Archived from the original on September 24, 2022. Retrieved September 19, 2022. Unknown parameter
|s2cid=ignored (help); Unknown parameter|url-status=ignored (help) - ↑ 24.0 24.1 Anzalone, Andrew (2020). "Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors". Nature Biotechnology. Nature. 38 (7): 824–844. doi:10.1038/s41587-020-0561-9. PMID 32572269 Check
|pmid=value (help). Archived from the original on October 9, 2022. Retrieved September 19, 2022. Unknown parameter|url-status=ignored (help); Unknown parameter|s2cid=ignored (help)
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