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Genome editing based on in vitro-assembled ribonucleoproteins in plants
2023 年 88 巻 4 号 p. 283-288
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2023 年 88 巻 4 号 p. 283-288
Gene editing technology based on the clustered regularly interspaced short palindromic repeats (CRISPR) system has garnered widespread use in plant genomes because of its cost-effectiveness, efficiency, and simplicity. To avoid the integration of foreign genes and any DNA fragments into target cell genomes, researchers have developed a system that introduces in vitro-assembled ribonucleoproteins (RNPs) consisting of guide RNA (gRNA) and Cas protein into target cells, enabling direct genome editing. This system was designed to deliver RNPs through four distinct methods: polyethylene glycol (PEG)-mediated cell transfection, particle bombardment, electroporation, and lipid transfection. In recent years, CRISPR technology has been extensively applied for the genetic modification of plants, providing a strategic response to environmental challenges. Researchers have successfully established RNP genome editing systems in various plant species. Despite some remaining issues, the RNP genome editing system still shows significant promise for future applications in the production of non-genetically modified (non-GM) crops.
Environmental stresses such as drought, extreme temperatures, and salinity, have been identified as significant factors leading to substantial agricultural production losses (Chen et al. 2019). In the 21st century, one of the primary challenges facing agriculture is improving crop yields through the accelerated development of resilient crop varieties (Kakoulidou et al. 2021). While conventional selection and breeding approaches, such as marker-assisted selection, have historically been employed to cultivate crop varieties, they are limited by their time-consuming nature and precision in genetic editing (Xiong et al. 2022). Gene editing technology represents a biotechnological breakthrough that enables precise genetic modifications in crops, significantly expediting variety development while mitigating the time constraints associated with traditional breeding methods. For example, targeted editing of genes related to drought tolerance in plants has shown promising results in enhancing plants’ resilience to drought stress (Li et al. 2022). In addition, gene editing technology can be applied to modify the metabolic pathways of medicinal or cash crops to increase the yields of bioactive compounds (Zhang et al. 2023).
Gene editing technology based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has gained widespread usage in plant genomes due to its affordability, efficiency, and simplicity (Zhang et al. 2019). The CRISPR system, an acquired immune system found in bacteria and archaea, combats viruses and bacteriophages through DNA recognition with CRISPR-RNA (crRNA) and DNA cleavage mediated by a nuclease known as CRISPR-associated proteins (Cas) (Ran et al. 2013). Guide RNA identifies gene sequences and guides Cas proteins which possess restriction endonuclease functions and bind specifically to target sites, resulting in DNA double-strand breaks (DSBs) and triggering endogenous DNA repair mechanisms (Kim 2018). The primary repair pathway in plant cells during DSBs is nonhomologous end joining (NHEJ), which can lead to the insertion and deletion of DNA fragments (Schmidt et al. 2019). The introduction of repair templates enables targeted foreign DNA integration through homology-directed repair (HDR) pathways (Puchta 2004, Schmidt et al. 2019). In addition to gene editing, the CRISPR system aids in visualizing a repetitive sequence or a specific gene locus (Fujimoto and Matsunaga 2016, 2017, Li and Matsunaga 2023).
Agrobacterium-mediated plasmid transformation is the most frequently utilized method for delivering the CRISPR system to plant cells. However, it carries the risk of integrating foreign genes into the genome of target cells, which raises safety concerns and limits its applicability in crop gene editing (Zhang et al. 2021a). Breeding can segregate foreign genes from edited target genes, but this process is typically time-consuming and labor-intensive, making it unsuitable for species with long growth cycles (Yang et al. 2023). Furthermore, if CRISPR genes are integrated into the target cell and continue to be expressed, the genomic DNA of target cell remains exposed to the CRISPR system, increasing the potential for off-target effects (Fu et al. 2013, Pattanayak et al. 2013). To address these concerns, researchers have developed a gene editing method that pre-assembles the two components of the CRISPR system, Cas protein, and guide RNA (gRNA), to form ribonucleoprotein (RNP) (Kim et al. 2014). This approach eliminates the need to introduce foreign genes into target cells for expression, enabling plants edited using RNP to be considered non-genetically modified (non-GM) plants. Additionally, RNP is rapidly degraded by intracellular proteases and nucleases, minimizing the potential for off-target effects resulting from prolonged exposure of genomic DNA to the CRISPR system (Fang et al. 2023).
The RNP complex primarily consists of the CRISPR endonuclease and gRNA. CRISPR endonuclease is a restricted endonuclease derived from archaea, and includes Cas9, Cas12a, Cas12b, Cas13, among others (Liu et al. 2022). Depending on the specific Cas proteins used, CRISPR-RNP complexes can be categorized into two major groups: the Cas9 system (Jiang et al. 2021) and the Cas12a system (Kim et al. 2017). The most prevalent system is the CRISPR/Cas9 system, in which gRNA guides the Cas9 protein to introduce site-specific DSBs, initiating a DNA repair mechanism predominantly mediated by NHEJ repair pathways. (Fig. 1, Cas9 system) This system is mainly used for imprecise gene editing, such as knockout (Subburaj et al. 2022, Poddar et al. 2023). Cas12a is another prominent Cas protein, distinct from Cas9, which recognizes PAM sequences different from the CRISPR/Cas9 system and generates sticky ends, facilitating HDR pathways for more precise editing (Fu et al. 2014, Kim et al. 2018). (Fig. 1, Cas12a system) These enzymes are suitable for editing different plant genes, thereby expanding the range of plant species amenable to genetic manipulation (Fang et al. 2023). Cas proteins can be synthesized using chemical methods, facilitating convenient in vitro RNP assembly (Zhang et al. 2021a).
RNP complex, composed of Cas protein and guide RNA (gRNA), can be categorized into two groups: the Cas9 system (of which gRNA comprises crRNA and tracrRNA, generate non-sticky ends) and the Cas12a system (which only requires crRNA and generate sticky ends). RNP complex can be introduced into protoplast cells for direct genome editing in three ways. In the nucleus, gRNA recognizes the target sequence and guides the Cas protein to cleave DNA to produce double-strand breaks that induce a DNA repair program. This technology can be applied to evaluate the gene editing efficiency of the CRISPR system and to breed non-GM crops that are resistant to environmental stresses including insect damage and drought stress.
The gRNA sequence identifies specific base sequences and guides Cas proteins to bind to them. Different CRISPR systems employ different gRNAs. In the CRISPR-Cas9 system, gRNA comprises CRISPR-RNA (crRNA) and trans-activating RNA (tracrRNA), with the target sequence located upstream of the protospacer adjacent motif (PAM) NGG. The Cas12a system only requires crRNA, located upstream of the 5’ end of the T-rich PAM sequence. Guide RNA can be synthesized through in vitro transcription (IVT) or chemical methods (Zhang et al. 2021a). The transcription template is first pre-processed with the T7 promoter before the gRNA sequence and subsequently transcribed in vitro by T7 RNA polymerase. Transcribed gRNA requires purification before RNP assembly (McGaw and Chong 2021). Research has demonstrated that IVT-produced gRNA may carry DNA contamination, which can integrate into the genome at the CRISPR cleavage site, leading to unwanted DNA insertions (Kim et al. 2017, Andersson et al. 2018). DNase treatment alone may not completely address this issue. In contrast, chemically synthesized gRNA exhibits high purity, eliminating concerns about DNA contamination (Andersson et al. 2018).
CRISPR restriction endonuclease and gRNA were mixed with a reaction buffer and incubated at room temperature for 10 min to assemble the RNP complex (Liang et al. 2017, Liu et al. 2020). Theoretically, a 1 : 1 radio of Cas protein to gRNA could be used for RNP assembly (Kim et al. 2014). However, in practical applications, a 3 : 1 or 1 : 1 radio of Cas protein to gRNA has been utilized for gene editing in apples (Osakabe et al. 2018), while a 1 : 3 ratio has been utilized for gene editing in petunias, soybeans, and tobacco (Malnoy et al., 2016, Subburaj et al. 2022).
There are four main methods for introducing RNP into plant cells: polyethylene glycol (PEG)-mediated cell transfection, particle bombardment, electroporation, and lipid transfection.
The PEG transfection method stands as the most prevalent approach for introducing RNPs into plant protoplasts, which are obtained following treatment with cellulase and pectinase enzymes the most prevalent approach for introducing RNPs obtained following treatment with cellulase and pectinase enzymes into plant protoplasts. PEG carries negative charges and can bind positively charged molecules on the cell membrane and affect the recognition function of the cell membrane (Robinson et al. 1979, Yoshihara et al. 2020), making it easier for RNP to enter cells. (Fig. 1, PEG-mediated cell transfection) This method serves as a rapid means of assessing the gene editing efficiency of the Cas system. PEG-mediated protoplast gene editing has been successfully adapted to numerous plant species, including Arabidopsis (Yoo et al. 2007, Woo et al. 2015), rice (Woo et al. 2015), apple (Malnoy et al. 2016), wheat (Liang et al. 2017), soybean (Kim et al. 2017, Kim et al. 2020), potato (Andersson et al. 2018, Carlsen et al. 2022), cabbage (Murovec et al. 2018), banana (Wu et al. 2020), Ben’s tobacco (Wu et al. 2023), Stevia rebaudiana (Ghose et al. 2022), chili pepper (Kim et al. 2020), and maize (Sant’Ana et al. 2020).
Particle bombardment, which is typically used to regenerate explants and plant tissues, is another commonly employed technique for delivering RNPs into plant cells. RNP-mediated genome editing via particle bombardment has been successfully demonstrated in rice (Banakar et al. 2020) and corn (Dong et al. 2021) as well as wheat (Liang et al. 2017, Poddar et al. 2023). Furthermore, electroporation and lipid transfection techniques can also be applied to introduce RNPs into recipient plant cells. Electroporation involves exposing cells to a strong electric field, rendering their membranes permeable to external molecules. (Fig. 1, Electroporation and Lipid transfection) This approach has been successfully adapted for gene editing in plants, including Chlamydomonas reinhardtii using CRISPR-Cas9 RNPs (Lee et al. 2020). Additionally, pre-assembled RNPs can be combined with cationic lipids to form liposomes, facilitating their fusion with negatively charged cell membranes for efficient RNP transfection (Yu et al. 2016, Liu et al. 2020). This method has been employed to deliver RNPs to tobacco plants (Liu et al. 2020).
Protoplasts are frequently chosen as targets for genome editing in plants due to their remarkable ability to readily incorporate foreign genes. These protoplasts are typically obtained by enzymatic hydrolyzing plant leaves, stem tips, or root tips with pectinase and cellulase enzymes (Nagata and Takebe 1970, 1971). Notably, protoplasts process robust regenerative potential and maintain cellular physiological activities like those of the whole plants. Genome editing performed on protoplasts allows for specific, rapid, and high-throughput analyses, as well as time-series experiments at the single-cell level, which aids in evaluating gene editing efficiencies and investigating of gene functions (Gilliard et al. 2021). However, it is important to acknowledge the limitations associated with protoplasts. Challenges related to their isolation, demanding culture conditions, and limited survival time must be taken into account (Gilliard et al. 2021).
In addition, gene-edited protoplasts generated using the CRISPR-RNP system can be regenerated to produce intact non-transgenic plants. However, the current method for protoplast regeneration is characterized by high genotype specificity and requires further advancement to enhance its applicability and success rate. It also imposes stringent requirements on culture conditions, including factors such as medium composition, temperature, and light (Reed and Bargmann 2021).
In recent years, CRISPR technology has been extensively applied to genetically modify plants, serving as a strategic response to environmental challenges. In the context of crop enhancement, researchers have successfully employed CRISPR-RNP technology to develop late blight resistant mutants in potatoes by targeting the susceptibility gene in protoplasts (Moon et al. 2022). Furthermore, CRISPR-RNP gene editing systems have been established in a range of crops. Including tobacco (Banakar et al. 2022a), chili peppers (Kim et al. 2020), rice (Banakar et al. 2020), tomatoes (Slaman et al. 2023), maize (Dong et al. 2021), and European chestnut (Pavese et al. 2022). In the future, the CRISPR-RNP gene editing system is expected to be widely employed in the genetic modification of crops to enhance adaptability to environmental stresses.
The CRISPR gene editing system is a notable method for rapid and highly efficient genome editing. By delivering RNPs directly into recipient plant cells for gene editing, it eliminates the integration of foreign genetic material into recipient cells. In fact, in some cases, the genome editing efficiency achieved using RNPs nearly equivalent to that of plasmid-based expression systems, enabling the production of non-transgenic gene-edited plants (Zhang et al. 2021b). This approach is becoming widely used in crop genetic enhancement. It is important to note that the efficiency of the CRISPR system depends on various factors, such as target genes, types of restriction endonucleases used, and transfection conditions. Different Cas9 proteins exhibit diverse activity and sequence specificity. Cas12a, for instance, has shown superior genome editing efficiency compared to Cas9 when delivered as RNPs in various plant species (Banakar et al. 2022b). Compared to the Streptococcus pyogenes Cas9 (SpCas9) system, Cas12a system uses short crRNA to mediate, and Cas12a protein possesses nuclease activity specifically targeted to cleave RNA, rendering it highly conducive for conducting multiplexed editing, making the Cas12a system more efficient for genome editing (Zhang et al. 2022). Research has also demonstrated that RNP delivery is less sensitive to temperature fluctuations compared to plasmid-based transformation systems, resulting in increased mutagenic efficiency during callus regeneration from protoplasts and the development of next-generation, recoverable heritable mutants (Banakar et al. 2022b). Additionally, the use of Trichostatin A (TSA) treatment has been shown to enhance the gene editing efficiency of RNPs in protoplasts (Choi et al. 2021). Furthermore, RNP-mediated genome editing technology has effectively regenerated non-GM plant individuals following the gene editing of protoplasts (Zhang et al. 2021a), providing new and broader possibilities for improving crops to adapt to the environment and increase yields.
This research was supported by a joint research funding from Yakult Pharmaceutical Industry Co., Ltd., MXT/JSPS KAKENHI funding to S. Matsunaga (20H05911 and 22H00415). It was also supported by JST-CREST (JPMJCR20S6), JST-OPERA (JPMJOP1832) and JST-GteX (JPMJGX23B0) grants to S. Matsunaga.
All authors contributed to the writing of this review.
