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Abstract
A critical step in generating gene‐edited plants is the removal of CRISPR‐related transgenes from T0 plants and their progenies, a process that is generally time‐consuming and labour‐intensive. We previously reported a Transgene Killer CRISPR (TKC) technology that enables self‐elimination of transgenes after the targeted gene has been edited. However, we observed that a small number of T1 plants generated by TKC still retained the transgenes. Herein, we first integrated
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Introduction
CRISPR-mediated gene editing has been widely used for genetic research and crop improvement in many plant species. Gene-editing technologies can introduce virtually any types of genetic modifications, including deletion, insertion, point mutation, transcriptional regulation and epigenetic manipulation (Gallego-Bartolomé et al., 2018; Gong et al., 2020; Hua et al., 2019; Huang et al., 2021; Jaganathan et al., 2018; Li et al., 2020b; Mao et al., 2019; Pan et al., 2021; Sedeek et al., 2019; Zhan et al., 2021; Zhang et al., 2017; Zhu et al., 2020). The main goal of plant gene editing is to generate stable, heritable and non-chimeric modifications in the genome so that the improved trait can be reliably maintained and transmitted to the following generations (Chen et al., 2019; Gao, 2021; Huang et al., 2021; Jiang et al., 2025; Mao et al., 2019; Zhang et al., 2019; Zhu et al., 2020). However, the continuous presence of CRISPR reagents or transgenes after the targeted gene has been edited poses several risks. The CRISPR transgenes increase the difficulty in assessing the phenotype and hereditability of the targeted modifications due to the random integration of transgenes in the genome that may affect the targeted trait for improvement (Gao et al., 2016; Gao and Zhao, 2014b), while also reducing the predictability of genetic analysis. The presence of gene-editing elements also increases the risk of off-target effects (Gao et al., 2016; Gao and Zhao, 2014b) and can lead to regulatory issues related to genetically modified organisms (Kim and Kim, 2016; Turnbull et al., 2021). Crops with any CRISPR transgene component residuals will unlikely receive approval for commercial applications from government regulatory agencies (El-Mounadi et al., 2020; Huang et al., 2016; Schmidt et al., 2020; Turnbull et al., 2021). Therefore, it is imperative to promptly remove the gene-editing elements once the targeted gene has been edited (He and Zhao, 2020; Metje-Sprink et al., 2018). Developing technologies that efficiently and quickly remove Cas9 and other transgenes after gene editing is completed will significantly facilitate both functional genomics and crop improvement using gene-editing technology.
Transgene-free genome editing technology is a potent approach for plant genetic improvement (He et al., 2022). For annual crops such as rice, identification of transgene-free edited plants by the traditional methods such as genetic segregation, backcross and genotyping is very laborious and time-consuming (Gao, 2021). Several approaches including exogenous or endogenous marker-assisted screening (Gao et al., 2016; Hoengenaert et al., 2025; Li et al., 2020a; Lu et al., 2025; Zou et al., 2025), viral delivery (Liu et al., 2023; Oh et al., 2025; Weiss et al., 2025; Wu et al., 2025) and some other methods have been widely used to isolate transgene-free plants (For a recent summary of the methods, see review articles (He et al., 2022; He and Zhao, 2020)). Among the methods, the Transgene Killer CRISPR (TKC) technology, which uses a pair of suicide cassettes to trigger self-elimination of the transgenes without compromising gene-editing efficiency (He et al., 2018, 2019; Liu et al., 2022; Yang et al., 2022), requires the least amount of hands-on time. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation. Similar to TKC, the pollen suicide strategy has also been adopted in other transgene-free gene-editing systems in rice and maize (Wang et al., 2023b; Yu et al., 2021, 2024).
However, we observed that the transgenes occasionally escape from TKC and a small number of T1 plants from T0 plants generated by TKC still contain transgenes (Liu et al., 2022). Previously, we developed a new reporter gene RUBY, which converts tyrosine into vivid red betalain, providing a visible marker without the need for special equipment or chemical treatments (He et al., 2020). RUBY is an effective selectable marker for identification of transformants in both monocots and dicots (Ge et al., 2023; Lee et al., 2023; Wang et al., 2023a; Wu et al., 2025; Zhao et al., 2022) and is particularly suitable for monitoring large crop plants under field conditions and for observing transformants and gene expression during tissue culture under sterile conditions. A gallery of RUBY plants is shown here ().
In this study, we used a single promoter to drive the expression of a synthetic gene that produces the guide RNA (gRNA), Cas9 and RUBY simultaneously. The gRNA unit was placed in an artificial tandem tRNA-gRNA-Ribozyme (inTGR) (Figure 1a). This RUBY-CRISPR architecture (Figure 1a) enables RUBY to act as a visible marker for transgenes and a proxy for Cas9 and gRNA expression, which has a strong correlation with gene-editing efficiency. Our design provides an easy and efficient way to select transgenic plants with a high confidence of targeted mutagenesis in the T0 generation. We further combined several TKC variants with the aforementioned RUBY-CRISPR unit to develop RUBY-TKCs as TKC version 2s (TKC2s) and performed gene editing in rice. We demonstrate that TKC2 systems developed here in our study provide an easy, rapid and highly effective strategy to generate transgene-free edited plants for either rice functional genomics or genetic improvement, with potential practical applications in genome editing of other agriculturally important crops, particularly polyploid crop species.
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Results
Coupling
To date, several approaches have been developed to express gRNAs driven by RNA polymerase II (Pol II) promoters, including Csy4 RNase from a bacterium (Nissim et al., 2014), self-cleavable ribozyme from viruses (Gao and Zhao, 2014a) and the endogenous tRNA processing enzymes (Xie et al., 2015). Recently, a synthetic gene encoding Cas9 protein with an intron containing a polycistronic tRNA-gRNA (PTG) was used for multiplex genome editing in rice (Ding et al., 2018). The intronic PTG (inPTG) utilized the endogenous mRNA splicing and tRNA processing machineries to generate several gRNAs and Cas9 from a single promoter.
We previously took advantages of the self-cleaving activities of ribozymes and developed an artificial gene named RGR (Ribozyme-gRNA-Ribozyme) that allowed us to produce gRNAs from both Pol II and Pol III promoters in various organisms (Gao and Zhao, 2014a; He et al., 2017). We further modified the above methods to produce gRNAs from an intron containing tRNA-gRNA-Ribozyme (inTGR) (Figure 1a). Additionally, we simplified this method by fusing the inTGR to the exon containing Cas9 and RUBY coding sequences as one synthetic gene (inTGR-Cas9-RUBY) (Figure 1a). Thus, gRNA, Cas9 and RUBY can be expressed from a single RNA Pol II promoter, allowing us to visually identify plants that have a high likelihood of being edited. We chose the Cestrum Yellow Leaf Curling Virus promoter (CmYLCV) fused with the CaMV 35S enhancer to express the synthetic gene (Figure 1a). The CmYLCV promoter drives comparable or higher levels of gene expression than the 35S promoter or maize (Zea mays) UBIQUITIN (ZmUbi) promoter in both dicots and monocots (Čermák et al., 2017; Stavolone et al., 2003). The CaMV35S-CmYLCV-U6 composite promoter notably enhanced the expression of the pegRNA and improved the prime-editing efficiencies in maize (Jiang et al., 2020). Leveraging these findings, we have successfully developed the RUBY-CRISPR plasmid through coupling RUBY with CRISPR.
When RUBY-CRISPR plasmid was transformed into rice calli through Agrobacterium-mediated transformation, we could clearly see that RUBY had varying degrees of accumulation in calli, roots, leaves and stalks, demonstrating that inTGR-Cas9-RUBY could be expressed from our constructs (Figure 1b). Furthermore, RUBY plants were easily differentiated from non-RUBY plants in fields, enabling selective seed harvesting (Figure 1b). RUBY colour was a good indicator of transgene-free efficiency. We used PCR with T-DNA-specific primers to detect the presence of transgenes in the T1 plants. 76.00% ± 7.57% of the RUBY-CRISPR T1 green plants (total 59 green plants in 208 T1 plants generated from 10 independent T0 transgenic plants) did not contain the CRISPR construct. For comparison, when a regular pCXUN-Cas9 construct was used, 20.57% ± 3.15% of T1 plants did not contain the CRISPR construct (Figure 1c, Tables S3 and S4).
Combining
We designed three types of suicide cassettes: microgamete-specific, endosperm-specific and zygote-specific. The microgamete-specific suicide cassettes include the pCaMV35S::Cytoplasmic Male Sterility 2 (CMS2) used in our previous study (He et al., 2018), and the maize α-amylase gene ZmAA1 with amyloplastic signal peptides under the pollen-specific promoter (Polygalacturonase 47, ZmPG47) (Allen and Lonsdale, 1993) kills transgene-containing pollens (Chang et al., 2016; Qi et al., 2020; Wu et al., 2016). The bacterial BARNASE gene (Mariani et al., 1990), under the control of the barley LTP2 promoter (Kalla et al., 1994) or the rice NF-YB1 promoter, was used to kill any endosperms that contain the transgene. We also used the zygote-specific suicide cassette, which has pREG2::BARNASE to kill transgenic zygotes (He et al., 2018, 2019).
We placed the suicide cassettes adjacent to the RUBY-CRISPR unit, resulting in the RUBY-TKC(TKC2) plasmids. Each RUBY-TKC plasmid contains two suicide transgene cassettes (Figure 2a). The p35S::CMS2 combined with pREG2::BARNASE was named RUBY-TKC1 (TKC2.1). The combination of pZmPG47::ZmAA1 and pREG2::BARNASE was named RUBY-TKC2 (TKC2.2). The pZmPG47::ZmAA1 combined with pLTP2::BARNASE was named RUBY-TKC3 (TKC2.3). The combination of pZmPG47::ZmAA1 and pNF-YB1::BARNASE was named RUBY-TKC4 (TKC2.4).
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To test the effectiveness and applicability of our TKC2 strategy, we targeted the rice PHOTOPERIOD SENSITIVITY5 (SE5) gene (Andrés et al., 2009) and the young seedling albino (YSA) gene (Su et al., 2012) (Figure 2b). The SE5 gene encodes an enzyme implicated in phytochrome chromophore biosynthesis. Loss-of-function se5 mutants show early flowering and a photoperiodic insensitivity phenotype (Andrés et al., 2009). The target sequence of the SE5 gene contained a SalI site near the PAM sequence. Small deletion/insertion at the target sequence would likely eliminate the restriction site, rendering SalI digestion resistance of the PCR products (Figure 2b). The YSA gene encodes a PPR protein with 16 tandem PPR motifs. Loss-of-function ysa mutants can develop albino leaves before the three-leaf stage, but the mutants gradually turn green and recover to normal green at the six-leaf stage (Su et al., 2012). The target sequence of the YSA gene contains an EagI site near the PAM sequence. Edited lines were verified via digestion of the PCR product with the restriction enzyme EagI (PCR-RE) and Sanger sequencing (Figure 2b).
The TKC2 plasmids were transformed into rice calli by Agrobacterium-mediated transformation. Subsequently, we detected that all T0 generation plants with RUBY colour were transgenic plants and had higher editing efficiency than green T0 generation transgenic plants (Figures 2c and S1a,b, Tables S1 and S2), indicating that RUBY colour was a good indicator of editing efficiency. In T0 plants targeting the SE5 gene (TKC2-SE5), the editing efficiency of green plants was 69.66% ± 11.64% while 89.61% ± 2.77% red plants were edited (Figures 2c and S1a, Table S1). In T0 plants targeting the YSA gene (TKC2-YSA), compared with the 67.84% ± 6.04% ysa mutation efficiency of green transgenic plants, 95.83% ± 3.61% of RUBY red transgenic plants were genome-edited mutants (Figures 2c and S1b, Table S2). Our results indicated that there was a strong correlation between RUBY expression and target gene editing, suggesting that RUBY can be used as a suitable indicator for determining transgenic mutant plants in the T0 generation and will not negatively affect plant genetic improvement. The visible phenotypes of se5 and ysa mutants allowed us to qualitatively evaluate the editing efficiency of our constructs. During the growth of T0 plants, we observed that the phenotypes of the se5 and ysa mutants (Figure 2d) were consistent with the previous results (Andrés et al., 2009; Su et al., 2012).
Visualisation of transgene escape from
Firstly, we calculated the ratio of transgene-free seedlings in T1 generation if not considering RUBY colour by using PCR detection with T-DNA-specific primers. We analysed the progenies of T1 plants from 33 independent TKC2-SE5 T0 plants that had the phenotypes of the se5 mutants. 99.44% ± 0.51% of the TKC2.1 T1 plants (total 98, from 6 independent T0 plants with se5 phenotypes) did not contain the CRISPR construct. For TKC2.2 T1 plants, 96.59% ± 1.30% (total 174, from 13 independent T0 plants with se5 phenotypes) were transgene-free (Figure 3a, Table S3). Furthermore, we observed more leakage of transgenes into T1 plants for TKC2.3 and TKC2.4. Only 80.01% ± 5.52% of the TKC2.3 T1 plants (total 95, from 5 independent T0 plants with se5 phenotypes) and 73.63% ± 4.30% of TKC2.4 plants did not contain the CRISPR construct (Figure 3a, Table S3). We also analysed the progenies from 28 independent T0 plants with ysa mutations in TKC2-YSA. We found that similar to TKC2-SE5, if not considering RUBY colour, the ratio without transgene elements was higher than 93.76% ± 2.09% in TKC2.1, TKC2.2 and TKC2.3 combinations (Figure 3a, Table S4). T1 plants (total 104, from 4 independent T0 plants with ysa phenotypes) of the TKC2.4 displayed a transgene-free ratio of 27.86% ± 2.83%, significantly lower than the 73.63% ± 4.30% of the TKC2.4-SE5 (Figure 3a, Tables S3 and S4), indicating that the transgenic scavenging ability of TKC-Unit4 was unstable.
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Some T1 plants from TKC2 T0 plants had red colour, indicating that transgenes were not eliminated in those plants by TKC (Figure S1c-l). Interestingly, we found that the ratios of green seedlings in T1 generation plants (Figure S2, Tables S3 and S4) were very similar to the ratios of transgene-free seedlings (Figure 3a, Tables S3 and S4) in T1 generation plants. This demonstrated that the RUBY colour could be used to assist in TKCs screening. Our results indicated that there were differences in transgene elimination ability among different combinations. For comparison, when using RUBY-CRISPR constructs, 26.65% ± 2.67% and 29.19% ± 4.03% of T1 plants derived from RUBY-CRISPR-SE5 and RUBY-CRISPR-YSA, respectively, lacked the CRISPR construct (Figure 3a, Tables S3 and S4).
Furthermore, when considering RUBY colour, we calculated the ratio of transgene-free seedlings in TKC2 T1 generation. We found RUBY is an effective visualisation of transgene escape in TKCs. TKCs in combination with RUBY improved the transgene elimination in T1 progenies. In T1 plants of TKC2-SE5, we analysed 473 T1 plants without RUBY colour (from 33 independent T0 plants). As shown in Figure 3b and Table S3, 100.00% of the TKC2.1 T1 plants (total 97, from 6 independent T0 plants with se5 phenotypes) were transgene-free. For all other TKC2 constructs, at least 96.00% of the T1 plants were transgene-free, demonstrating that our TKC2 strategy was very effective in eliminating the transgenes. Remarkably, in the progenies of TKC2-YSA, we further analysed all of the 382 T1 plants without RUBY colour (from 28 independent T0 plants with ysa phenotypes) and found that the ratio without transgene elements was 100.00% in all four TKC2 combinations (Figure 3b, Table S4). These results indicate that the RUBY colour was very effective for spotting the plants in which transgenes escaped from TKCs.
Analyses of mutations in the target gene generated by
The visible phenotypes of se5 and ysa mutants allowed us to qualitatively evaluate the editing efficiency of our constructs. During the growth of T1 plants, we observed that the phenotypes of the se5 and ysa mutants (Figure 3c) were consistent with the previous results (Andrés et al., 2009; Su et al., 2012). When analysing the T1 mutants of TKC2s, we found 95.42% ~ 100.00% T1 green mutants of four TKC2-SE5 did not contain the CRISPR construct in 337 T1 green mutants derived from 33 independent T0 plants with se5 mutations (Table S3). Meanwhile, it was surprising that we found that all of the T1 green mutants of four TKC2-YSA did not contain the CRISPR construct in 339 T1 green mutants from 28 independent T0 plants with ysa phenotypes (Table S4), demonstrating that our TKC2 strategy was very effective in generating transgene-free mutants.
Furthermore, we analysed the green progenies of the 33 and 28 independent T0 plants with se5 and ysa mutations, respectively. Most of the edited mutations were stably inherited in the subsequent generations normally (Figures 3d, S3 and S5). However, there were some interesting segregation patterns (Figures 3d, S3 and S4). In TKC2-SE5, progenies from the T0 plant #2-SE5-2 had three types of alleles: a deletion of 21 base pairs, a wild type and a deletion of ‘CG’, suggesting that the #2-SE5-2 T0 plant was mosaic (Figure 3d). Similar mosaicism was also observed among the T1 plants from the T0 plant #2-SE5-13 and #2-SE5-23 (Figure S3). Without eliminating the CRISPR construct, it would be much more difficult to interpret that these mutations were inherited from their T0 plants. Furthermore, we observed that two transgene-positive progenies of T0 plant #2-SE5-7 displayed genotypes of +G/−3/WT/−1/−5/−4 and −21/+g/−3/−1, respectively, indicating that these two T1 plants were chimeras (Figure S4), which were newly created by Cas9 in the T1 generation. Our findings demonstrate that the presence of Cas9 is detrimental to the stable inheritance of mutations. We also observed some complex segregation patterns of the T1 lines from the T0 plants in TKC2-YSA (Figures 3d and S5). The mosaic nature of T0 plants was obvious for the plant #1-YSA-18, which produced three different alleles: a deletion of ‘GG’, an insertion of ‘A’ and a wild type. In addition, the ‘GG’ deletion was more prominent. It would be very difficult to determine whether the second and third alleles were newly generated by Cas9 or inherited from T0 if the CRISPR construct were not eliminated.
Whole genome sequencing of
To further verify our results, which were based on the digestion of PCR product with restriction enzymes (PCR-RE) and Sanger sequencing, 24 transgene-free green mutants, four transgenic green mutants and four transgenic RUBY mutants in the T1 generation of the four TKC2 combinations were selected for whole genome sequencing (WGS) (Table S5). Mapping analysis indicated that the DNA constructs were integrated into the genomes of the transgenic RUBY mutants #3-SE5-29-28, #1-YSA-14-7, #3-YSA-3-1 and #4-YSA-10-1, respectively, as determined by the presence of construct reads (Figures 4 and S6). By contrast, genomic analysis of 24 transgene-free green mutants revealed no fragments of the T-DNA constructs, except the rice endogenous sequences and some mismatches, as evidenced by visual analysis of the construct fragments using IGV ver. 2.16.2 (Figures 4 and S6). WGS mapping analysis showed that the putative transgenic green mutants #3-SE5-8-2, #4-SE5-17-6 and #4-SE5-17-31 were indeed transgenic with integrated CRISPR construct. The line #4-SE5-3-17 was transgene-free, which may have been caused by false positives during the PCR process (Figures 4c,d and S6d). Together, these results indicated that the transgene components were successfully eliminated by TKC2 from the rice genome.
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In parallel, WGS analysis confirmed editing of the target genes SE5 and YSA (Figure S7). Interestingly, two transgenic lines #3-SE5-29-28 and #4-SE5-17-31 harboured three or more genotypes at the target site, suggesting that these two lines were chimeras (Figure S8). This is newly created by Cas9, which also demonstrates that the presence of Cas9 is not conducive to the stable inheritance of mutations.
Discussion
In this paper, we combined RUBY and TKCs to enrich target gene-edited rice plants in T0 generation and to effectively eliminate transgenes in target gene-edited rice plants in T1 generation. We used a single cassette to express gRNA, Cas9 and RUBY, so that we could use the visible marker RUBY as a proxy for the expression levels of gRNA and Cas9. RUBY makes it easy to select transgenic plants in the T0 generation. RUBY also makes it effortless to spot transgene escape from the TKC system (Figure 5, Tables S6 and S7). In our previous study, we used antibiotics to screen the candidate genome-edited plants and found that some plants regenerated from antibiotic-resistant calli were not transgenic plants (He et al., 2017). In this study, we found that all of the RUBY plants were transgenic plants and that most RUBY plants had higher editing efficiency at the target locus than green plants (Figures 2c and S1a,b, Tables S1 and S2). Our results suggest that the extent of RUBY colour accumulation might be indicative of the expression levels of other linked transgenes such as Cas9 and gRNA, thus providing a robust and speedy method for identifying plants with elevated gene-editing efficiency.
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In plant genome editing, incomplete editing frequently occurs in the T0 generation. When the T1 plants are homozygous or exhibit bi-allelic mutations, we could infer that the target gene in the corresponding T0 plant was completely edited. In such cases, if the gene-editing components are present, the mutations in the T1 plants will not change. However, we observed that a considerable proportion of T1 families still contained plants with wild-type or heterozygous genotypes, indicating that the target sites were either not edited or incompletely edited (Figures 3d and S3). Our results suggest that the target sites in these corresponding T0 plants were incompletely edited. In such situations, if the gene-editing elements are still in the plants, the unedited target sites in the T1 plants are likely to undergo further editing, leading to the generation of new genotypes (Figures S4 and S8). This necessitates the resequencing of T1 plants to confirm their mutations, which increases the workload and cost of sequencing. Moreover, we found that in certain T1 families without transgene residues, three haplotypes were present (Figures 3d and S3), such as the #2-SE5-2 family, which exhibited three haplotypes: −21 bp, −2 bp and WT (Figure 3d). This suggests that the corresponding T0 plant was a chimeric individual containing three haplotypes. Since rice is a diploid plant, a gene typically has no more than two haplotypes. If the T1 plant has not been eliminated of the gene-editing components, we could not determine whether the third haplotype was inherited from the T0 generation or newly generated in the T1 generation, thus preventing us from confirming whether the T0 plant was a chimeric individual. Therefore, using the updated TKC technology for genome editing allows for more stable mutation identification in T0 or T1 plants. Even if the T0 plant is incompletely edited, the mutations detected in the T0 plant can represent those in the T1 plant, eliminating the need for repeated sequencing to confirm mutations. Furthermore, this technology provides strong support for determining whether the T0 plant is a chimeric individual.
Transgene-free lines without T-DNA eliminate the potential disruption of gene functions at the insertion site caused by T-DNA in Agrobacterium-mediated stable transformation (Gao and Zhao, 2013; O'Malley and Ecker, 2010). We would like to point out that any DNA-based method can potentially leave small fragments of foreign DNA in the genome. It is imperative to analyse multiple alleles resulting from independent transformation events in order to rule out the contributions from such random insertions (He et al., 2022; Liu et al., 2021; Wu et al., 2022). Likewise, it would be prudent to sequence the whole genome of the genome-edited plants before commercially growing. WGS data indicate that our updated TKC2 technique can completely eliminate transgene residues, resulting in gene-edited plants free of any transgenic fragments.
In conclusion, we have developed a genome editing system to efficiently isolate transgene-free, genome-edited plants. Our technology does not require PCR-based transgene genotyping in both T0 and subsequent generations. Combining RUBY with TKCs greatly improved the efficiency in obtaining transgene-free genome-edited rice plants. The four new vectors presented here provided more options and flexibility for scientists to use our TKC2 technology (Figure 5). Using RUBY as a reporter to assist TKCs has less environmental and health concerns compared with antibiotic and/or herbicide resistance markers. It is easy to use and does not add more steps when constructing a TKC2 vector than the traditional CRISPR vectors. RUBY has been widely applied in numerous plant species (Li et al., 2023; Tse et al., 2024; Yi et al., 2024). Therefore, this technology could be readily applied to other CRISPR/Cas systems and other sexually reproduced crops to generate transgene-free, genome-edited plants in the T1 generation. Moreover, this technology prevents the release of transgenic pollen or seeds into the environment, which can effectively alleviate serious concerns regarding the widespread spread of transgenic elements into natural and agricultural ecosystems. This is friendly to the new regulations of new biotechnological plants around the world (Buchholzer and Frommer, 2023; Sprink et al., 2022). It is expected that TKC2 has broad applications in plant genetic studies and crop genetic improvements.
Materials and methods
Rice materials
Rice cultivar cv Zhonghua 11 [Oryza sativa L. ssp. geng (japonica)] was used for the study. Rice plants were grown in a greenhouse in winter and normal fields under natural conditions in summer in Nanjing City, Jiangsu Province, China.
Construction of the
The RUBY-CRISPR plasmids were constructed using the CRISPR vector pHEE401-mCherry as the backbone (Mudgett et al., 2023). We first cloned the RUBY ORF and the HSP18.2 terminator, which were amplified from the Actin1: RUBY vector (He et al., 2020). The RUBY, along with the HSP terminator, was cloned into pHEE401-mCherry by Gibson assembly between the KpnI and EcoRI sites (Gibson et al., 2009), using the primers RUBY-F and RUBY-R, resulting in the plasmid 401-01. We took advantage of the P2A peptide, which has the sequence of (GSGATNFSLLKQAGDVEENPGP), to link the Cas9 and RUBY coding regions. The 2A peptide was cloned into 401-01 by Gibson assembly between the SacI and Bsp119I sites (Gibson et al., 2009), using the primers P2A-F and P2A-R, resulting in the plasmid 401-04.
The promoter of Cas9 in 401-04 was replaced with the 35S-CmYLCV promoter that was synthesized by TSINGKE (Nanjing, China) at the SpeI and XbaI sites using the primers 35S-CmYLCV-F and 35S-CmYLCV-R. The resulting plasmid was called 401-05. The intron-tRNA-SpeI-gRNA-HDV-intron (inTGR) fragment was synthesized commercially (TSINGKE, Nanjing, China) and then was inserted into the XbaI-digested 401-05 vector using the primers intron-F and intron-R, generating the plasmids RUBY-CRISPR.
Insertion of
We prepared several DNA units for constructing the TKC plasmids. In TKC2, TKC3 and TKC4, the PG47 promoter was amplified from the genomic DNA of maize cultivar B73 using the primers ZmPG47-F and ZmPG47-R; the ZmAA1 gene was synthesized from Beijing Tsingke Biotech and then amplified by the primer pair ZmAA1-F and ZmAA1-R. In TKC3, the Ltp2 promoter DNA was synthesised from Beijing Tsingke Biotech and then amplified using the primers HvLtp2-F and HvLtp2-R. In TKC4, the NF-YB promoter was amplified from the genomic DNA of rice cultivar Zhonghua 11 (ZH11) using the primers NFYB-F and NFYB-R. The 35S-CMS2-Nost and REG2-BARNASE expression cassettes were present in the TKC plasmid (He et al., 2018). The primers used in this study were listed in Table S8.
The RUBY-TKC plasmids were built on the basis of the RUBY-CRISPR. The other cassettes were amplified by PCR and cloned into RUBY-CRISPR using Gibson assembly (Gibson et al., 2009). In TKC1, we used the TKC plasmid (He et al., 2018) as templates to construct the 35S-CMS2-Nost expression unit with the primer pairs 35S-CMS2-Nost-F/35S-CMS2-Nost-R. The PCR product was inserted into the NcoI site in RUBY-CRISPR by Gibson assembly to produce the plasmid 35S-CMS2-RUBY. In TKC2, TKC3 and TKC4, we constructed the PG47-ZmAA1-Nost expression unit by overlapping PCR reactions and cloned the unit into the NcoI site in RUBY-CRISPR. The resulting plasmid was called PG47-ZmAA1-RUBY.
We constructed the REG2-BARNASE-tAtAdh expression unit by overlapping PCR reactions and cloned the unit into the HindIII site in 35S-CMS2-RUBY and PG47-ZmAA1-RUBY, respectively. The resulting plasmid was called RUBY-TKC1 and RUBY-TKC2. We also used overlapping PCR reactions to generate the Ltp2-BARNASE-tAtAdh expression cassette and the NFYB-BARNASE-tAtAdh expression cassette, which were cloned into the HindIII site of the plasmid PG47-ZmAA1-RUBY, respectively. The resulting plasmids were called RUBY-TKC3 and RUBY-TKC4. The primers used during these construction steps are listed in Table S8.
Insertion of
We generated plasmids RUBY-TKC-SE5 by introducing the SE5 target sequence into the SpeI site using the primers intron-SE5-F and intron-SE5-R, which produced a guide RNA to target the rice SE5 gene. The target sequence was designed by a web tool CRISPR-P (Lei et al., 2014). We also constructed RUBY-TKC-YSA plasmids, which targeted the YSA gene. The target sequences of SE5 and YSA were shown in Figure 2B. The primers used in this study were listed in Table S8.
Plant transformation
The RUBY-TKC-SE5 and RUBY-TKC-YSA plasmids were transformed into calli of ZH11 through Agrobacteria-mediated plant transformation following a protocol that was previously described (Hiei et al., 1994). All plasmids were introduced into Agrobacterium tumefaciens strain EHA105 using electroporation (Meng et al., 2017). T0 plants were visually scored for RUBY phenotype at the calli culture stage and at different growing stages in the natural field. Seeds from each individual T0 plant were harvested separately.
Transgene-positive detection and mutation identification
We selected all T0 plants exhibiting RUBY colour to determine the efficiency of RUBY in selecting the transgene plants. The templates were extracted from the leaves of rice plants. The primer pair TG-F/TG-R was used to amplify part of the RUBY fragment of RUBY-TKC plasmids in rice. We used the genomic DNA from ZH11 as negative controls. The quality of rice genome DNA was tested by primer pair RG-F/RG-R.
We randomly selected the T1 progenies from 33 independent T0 transgene plants of RUBY-TKC-SE5 and 28 independent RUBY-TKC-YSA T0 transgene plants to determine the efficiency of RUBY-TKC systems in self-eliminating the transgenes and in editing the target genes. The primer pair TG-F/TG-R was used to amplify the T-DNA fragment of the RUBY-TKC plasmids in rice. We used the genomic DNA from the corresponding T0 plants as positive controls and DNA from ZH11 as negative controls. The quality of rice genome DNA was tested by the primer pair RG-F/RG-R.
We used SE5-GT1/SE5-GT2 to amplify part of the SE5 gene from the RUBY-TKC-SE5 T0 plants and the green T1 plants we analysed. PCR products were digested with SalI. We also used primer pair YSA-GT1/YSA-GT2 to amplify part of the YSA gene from the RUBY-TKC-YSA T0 plants and the green T1 plants. PCR products were digested with EagI. The existence of a SalI and EagI resistant band was a clear indication of a successful editing events. We also directly sequenced some of the PCR products by using primers SE5-GT1 and YSA-GT1, respectively. For heterozygous or bi-allelic plants, the overlapping peaks were resolved using the publicly available Dsdecode site () (Xie et al., 2017) and ICE site () (Conant et al., 2022). All primers used for genotype analysis were listed in Table S8.
Whole genome sequencing and data analysis
The 150 bp paired-end reads whole genome sequencing data were generated using the Illumina NovaSeq 6000 platform by WHbioacme. At least 8.15Gb of sequencing data were obtained for each sample, ensuring sufficient coverage for subsequent genomic analysis. The raw reads were filtered using Fastp version 0.23.4 to remove low-quality reads (Chen et al., 2018). The high-quality paired-end short genomic reads were mapped to the reference genomes of rice (MSU7.0) using BWA software version 0.7.17 (Li and Durbin, 2009). The mutations (SNPs, deletions and insertions) in the gene-edited plant genomes were generated using the SAMtools package version 1.19 (Danecek et al., 2021). The mutations were filtered on the basis of quality and sequence depth, and the target site mutations were visualised using IGV software version 2.16.0 (Robinson et al., 2011). To determine whether the edited lines contain transgenes or fragments from the constructs, the high-quality paired-end short genomic reads were mapped to the constructs and T-DNA plasmid sequences using BWA software version 0.7.17.
Acknowledgements
We apologize to those whose work we were unable to cite due to space and reference limitations. We are deeply grateful to Prof. Yunde Zhao from the Department of Cell and Developmental Biology, University California San Diego, and Prof. Lanqin Xia from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, for their insightful comments and suggestions. This work is partly supported by the National Natural Science Foundation of China (32200335 and U21A20207), the Youth Innovation Program of the Chinese Academy of Agricultural Sciences (Y2023QC39), the Nanfan special project of CAAS (YBXM2405 and YBXM2446), the Hainan Seed Industry Laboratory (Grant No. B23CJ0208) and the Earmarked Fund for China Agriculture Research System (CARS-01).
Author contributions
M.Z. and Y.H. conceived the idea and wrote the first draft of the manuscript. M.Z., L.Y., Z.Z., H.C., D.W., M.X., Z.Z. and Y.Z. conducted the experiments. M.Z. analysed the data. N.Y., J.W., H.Z., Y.T. and L.X. contributed to manuscript revision. Y.H. supervised the study and revised the manuscript. All authors contributed to the article and approved the submitted version.
Conflicts of interest
The authors declare no conflicts of interest.
Data availability statement
The WGS sequence data reported in this study have been deposited in the National Center for Biotechnology Information (NCBI) BioProject database under accession number PRJNA1254482.
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