Highly efficient transgene-free plant genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR

doi:10.21203/rs.3.rs-1637574/v1

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Highly efficient transgene-free plant genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR

https://doi.org/10.21203/rs.3.rs-1637574/v1

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Background: CRISPR-Cas9 Genome-editing technology has revolutionized the plant science and hold enormous promise in crop improvement. When performing the gene editing system for crops, it is crucial to eliminate of the Cas9/sgRNA T-DNA cassette in the T1 generation.

Results: Here, we firstly validated that incorporating an OsU3-tRNA promoter combination in the CRISPR/Cas9 system contributed to the highest mutagenesis efficiency that increased sgRNA expression levels over the AtU6-tRNA and AtU6 promoters by editing the NtPDS gene (Ntab0595110) in tobacco. Then we optimized the existing tobacco CRISPR/Cas9 system by using the OsU3-tRNA promoter combination instead of AtU6, and fusing an AtUb10-Ros1 expression cassette in T-DNA for monitoring the transgene events. The new vector was named as pOREU3TR. As expected, 52 transgene-free and homozygous gene-edited green plants were effectively screened at T₁ generation by editing the NtLHT1 gene (Ntab0818090) in tobacco, and the contents of most free amino acids in the T₂ mutants ntlht1 leaves were detected significantly different from those in the wild type leaves, demonstrating the highly efficient of the system.

Conclusions: This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements to increase the efficiency of plant genome editing.

CRISPR/Cas9

OsU3-tRNA

transgene-free

Ros1

NtLHT1

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has emerged and widely applied in recent years as a robust technology for genome editing in various organisms. It is a valuable tool in creating new materials for researches. And the editing efficiency of the system is closely related to the expression level of the single guide RNA (sgRNA) [1–4]. It is generally believed that the RNA polymerase III (pol III) promoters are commonly used in CRISPR/Cas9 vectors for driving sgRNA expression in plants, such as Arabidopsis U6 (AtU6) and AtU3 promoters often preferred in dicotyledons, whereas the rice U6 (OsU6) and OsU3 promoters in monocotyledons. U6 snRNAs play their roles in the intron splicing of pre-mRNA in the nucleus, while U3 snRNAs participate in pre-rRNA processing [5–7]. The OsU3 exhibits high editing efficiency in rice and can be transcribed normally in dicot plants [8, 9], indicating that the OsU3 promoter also could be applied to gene editing of dicotyledons. The transfer RNAs (tRNA) are involved in the protein translation. Because of the internal transcriptional elements which can recruit pol III complexes and the endogenous tRNA-processing system, it has been reported that the tRNA-sgRNA architecture played a technical function in boosting the editing capability [10–13]. Based on these features, we designed two promoter combinations AtU6-tRNA and OsU3-tRNA to drive the expression of sgRNA targeting the NtPDS gene in tobacco respectively.

After transformation of the tobacco callus, the follow missions are to select the active transformants with expected mutations in T₀ transgenes and identify the Cas9/sgRNA T-DNA free mutants in T1 generation. Enzyme digestion or sequencing of the PCR amplicons which were usually performed in the isolation are both time-consuming and laborious, therefore, Cas9/sgRNA expression levels are often monitored indirectly. The application of fluorescent genes coupled with Cas9/sgRNA expression units have considerably contributed to an approach for identifying plants with elevated Cas9/sgRNA concentrations in transgenic progenies. But the method is usually limited by the special assistant instruments such as fluorescent microscope[14, 15]. Nowadays, other creative genes coupling with Cas9/sgRNA cassettes show visible phenotypes when they are edited, which were explored and applied more effectively, such as the genes involved in trichome development and anthocyanins biosynthesis[16–18].

The Antirrhinum Rosea1 (Ros1) is an MYB-type transcription factor gene activating the biosynthesis of anthocyanin pigments. The Studies reported that the tobacco transgenic plants overexpressing Ros1 displayed dark red pigmentation by the naked eye in leaves and flowers which provided a visible marker in tobacco. Meanwhile the abiotic stress tolerance also could be enhanced by the gene[19, 20]. Here we optimized the detection efficiency by coupling of Ros1 under the control of the Arabidopsis Ubiqutin 10 (AtUb10) promoter leads to the accumulation of anthocyanin in leaves and flowers, providing a visible marker for selecting the needed transgenic plants.

Amino acids in tobacco are not only the major nutrients essential for plants growth and development, but also the important substances related to the quality and flavor of tobacco leaves[21]. The absorption and transport of them depend on the transport function of amino acid transporters (AATs). LHT1 is a member of the lysine/histidine transporters (LHTs) family, an important subfamily of amino acid transporters. It was initially viewed as a lysine and histidine selective transporter which was determined in yeast, while, subsequent studies revealed that LHT1 could transport a broad spectrum of amino acids with preference for neutral and acidic amino acids[22]. LHT1 gene was mainly expressed in roots and leaves. The gene could mediate not only amino acids uptake from soil, but also translocation and partitioning of amino acids within plants[23]. However, it should be noted that the research on LHT1 was mainly performed in Arabidopsis, while our understanding of LHT1 in tobacco is generally scant.

In the present study, we firstly constructed AtU6-tRNA and OsU3-tRNA promoter combinations to drive the expression of sgRNA respectively and investigated their efficacy and efficiency in genome editing by targeting the tobacco phytoene desaturase (NtPDS) gene (Ntab0595110), which was used as a phenotypic marker for causing plant bleaching when the gene was silenced [24]. The results showed that the use of OsU3-tRNA promoter combination achieved the highest editing efficiency in tobacco cells. We then optimized the CRISPR/Cas9 system using the OsU3-tRNA promoter combination instead of the AtU6, and added an AtUb10-Ros1 expression cassette next to the Cas9/sgRNA units in the plasmid. The editing of the tobacco NtLHT1 gene (Ntab0818090) demonstrated the efficacy of the system, and the contents of the free amino acids in the mutant leaves were significantly different from those in wild type leaves. Additionally, the successful editing of the tobacco NtLHT1 gene suggests that the OsU3-tRNA-sgRNA and AtUb10-Ros1 system might be applied in other monocot and dicot plants.

OsU3-tRNA promoter combination enhanced the editing efficiency

It has been reported that the rice OsU3 can be transcribed normally in dicot plants and the tRNA-sgRNA architecture played a technical function in boosting the editing capability [8, 10, 11]. To determine whether the use of AtU6-tRNA and OsU3-tRNA promoter combinations results in improved genome editing efficiency in tobacco, we constructed CRISPR/Cas9 vectors based on the backbone of the pORE-Cas9 vector using the OsU3-tRNA/AtU6-tRNA/AtU6 promoters to express PDS sgRNA respectively (Fig. 1a). The CRISPR vectors were designated OsU3-tRNA-PDS, AtU6-tRNA-PDS and AtU6-PDS. The vectors were introduced into tobacco leaf disk via Agrobacterium-mediated transformation. Neomycin-resistant callus were obtained after antibiotic-dependent selection and albino callus were obtained as expected (Fig. 1b). Exogenous T-DNA insertions were identified by PCR using Cas9-specific primers (Supplementary Table S1). The results revealed that all the neomycin-resistant calluses contained the exogenous Cas9 gene (Fig. 1c), which suggested that the CRISPR vectors had been successfully introduced into the tobacco cells. We then checked the target site of the PDS gene in these transgenic calluses via PCR approach. The desired bands were purified and sent for Sanger sequencing. As expected, the indel (deletions or insertions) mutations at the target site were observed in the transgenic callus. The mutation rates of the callus for OsU3-tRNA-PDS, AtU6-tRNA-PDS and AtU6-PDS were 80%, 65.7% and 60.7% respectively (Fig. 1d). The expression of PDS sgRNA was detected by quantitative real-time PCR (qRT-PCR). According to the results, the AtU6, OsU3-tRNA and AtU6-tRNA promoters successfully promoted the expression of PDS sgRNA in tobacco leaves, and the sgRNA expression levels driven by the OsU3-tRNA and AtU6-tRNA promoters were higher than those coordinated by the AtU6 promoter (Fig. 1e). These results demonstrated the efficacy of the OsU3-tRNA, AtU6-tRNA, and AtU6 promoters in CRISPR/Cas9-mediated genome editing in tobacco.

According to the sequencing results of the PDS gene in T₁ plants, the mutation rates of OsU3-tRNA-PDS (88.5%, 23/26) and AtU6-tRNA-PDS (80.9%, 17/21) were higher than that of AtU6-PDS (64.7%, 22/34). The possible reason is that the tRNA could enhance the PDS sgRNA expression and play a role in sgRNA caching and maturation. The homozygote plants were obtained with the OsU3-tRNA and AtU6-tRNA promoter combinations at rates of 19.2% and 9.5% respectively (Table 1). Collectively, these results suggested that the use of OsU3-tRNA promoter combination resulted in high editing efficiency in tobacco.

Table 1

Overview of site-specific editing for PDS gene using different promoters
Vectors	Number of plants	Number of mutations	Number of homozygotes	Mutation rate (%)	Homozygotes rate (%)
OsU3-tRNA-PDS	26	23	5	88.5	19.2
AtU6-tRNA-PDS	21	17	2	85.7	9.5
AtU6-PDS	34	22	0	64.7	0
Supplementary Table S1 Primers used in this study

Ros1 Improved The Screening Efficiency Of The Transgene-free And Gene-edited Plants

In order to obtain stably transmissible mutations in tobacco generated by CRISPR/Cas9-mediated genome editing technology, it is necessary to segregate out the CRISPR/Cas9 construct. However, it is laborious and inefficient to isolate the Cas9-free mutants in the transforming generations by the traditional PCR method. Here, we inserted a dark red tag (Ros1) driven by the AtUb10 promoter into OsU3-tRNA to obtain a visual screen of mutants (Fig. 2). This modified vector was named pOREU3TR and tested by editing the LHT1 gene in tobacco. When plasmids containing the pOREU3TR-LHT1 unit were transformed into tobacco, we observed that anthocyanin accumulated in callus, leaves, stem, root and the flowers (Fig. 3a and b), producing a visible dark red color, which can facilitate the detection of transgenic events in callus and plants.

We found that all of the T₀ plants that displayed dark red color contained the Cas9 expression cassette revealed by our PCR analyses (Supplementary Fig. 1a) and were then detected out with different indels mutations in the target site, including heterozygous and homozygous indels mutations through the Sanger sequencing (Supplementary Fig. 1b). The dark red T₀ lines with homozygous mutations were screened to transplant from medium into soil in a climate-controlled growth chamber and selfed to obtain the seeds for T₁ population.

Approximately 500 T₁ plants from each single homozygous LHT1 gene-edited transgenic T₀ tobacco lines were grown. In contrast to the all-purple-red phenotype of the T₀ transgenic plants, the T1 segregants of them had either dark red or green phenotypes (Fig. 3b). We hypothesized the green ones (52) were our needed plants without the T-DNA. To detect whether the transgenes were present in the green T1 plants, DNA was extracted from each green plant and tested using Cas9 primers. As shown in Fig. 3c, d and e, all green T₁ plants did not contain the transgene elements, and sequencing results showed that they had the same homozygous mutation types in the target site with the parent, demonstrating that our CRISPR/Cas9 strategy was highly effective for identifying transgene-free and gene-edited T₁ plants.

Knockout of NtLHT1 affected the contents of amino acids in leaves and plant height

The green transgene-free T₁ plants containing a homozygous mutation resulting in premature stop codons were allowed to self-fertilize for the further analysis in the T₂ generation. To investigate the effect of the knockout of NtLHT1 gene on tobacco, the T₂ ntlht1 mutants and WT plants were planted in soil in the greenhouse. The plant height and amino acids content in the leaves of each plant at different stages were further determined by direct measuring and HPLC analysis respectively. The results showed that, compared with the wild type, the plant height was reduced significantly in the ntlht1 plants at the maturity stage (Fig. 4d). Additionally, the amounts of most amino acids in ntlht1 leaves were significantly less at rosette stage and flower-bud appearing stage, while significantly higher at maturity stage than those in the wild type plants, including Asp、Ser、Gly、Arg、Pro、Met、Leu and Total amino acids contents (Fig. 4a, b and c). These results indicated that a lack of NtLHT1 function affected tobacco normal growth, and the transport and distribution of amino acids in leaves.

As an RNA-guided DNA endonuclease system, sgRNA activity and the expression of sgRNA greatly influence the efficiency of CRISPR/Cas9-mediated genome editing [25]. To date, the widely used eukaryotic U3 and U6 promoters in monocot and dicot plants have been isolated from rice and Arabidopsis, respectively [26]. The application of tRNA is an effective strategy for improving the efficiency of genome engineering in plants [12]. In this study, we successfully used OsU3-tRNA promoter combination to regulate the expression of PDS sgRNA and LHT1 sgRNA in the dicot tobacco, reaching a higher mutation rate than AtU6 and AtU6-tRNA (Fig. 1), which is unexpected. The possible reason is that the internal promoter elements of tRNA which can recruit the Pol-III complex compensated some function of rice OsU3 promoter in a dicot plant [10, 12]. The results suggested that the use of OsU3-tRNA promoter combination could increase the genome editing efficiency and be applied in both monocot and dicot plants.

Despite the several reports describing a visible role for MYB TFs genes in optimizing the CRISPR/Cas9 system, few studies have reported the MYB TF Ros1 applied in improving the system [17, 27]. Some previous studies have determined that Ros1 overexpression in tobacco, as well as in snapdragon flowers, petunia and tomato enhanced anthocyanin accumulation in the whole vegetative and floral tissues by elevating the transcription of all key genes involved in the biosynthesis of this pigment [19]. In this study, we constructed an effective CRISPR/Cas9 system, pOREU3TR, including the Ros1 expression module. The results showed that all the transgene callus and plants exhibited visible dark red phenotype, making the screening procedure easily and efficiently, especially in the T₁ generation with only 10.5% (52/495) plants were green (Fig. 3d). These demonstrated that the Ros1 gene could be well applied in the gene editing system.

AtLHT1, which is the first member identified in the LHT subfamily, was regarded as a Lys and His selective transporter for transporting Lys and His most efficiently among the 13 different amino acids tested in the uptake measurements in yeast [28]. However, given the substrate specificity of the LHTs identified so far, it could be confirmed that the LHTs were not Lys and His unique transporters, but ones with broad substrate specificity and with preference for neutral and acidic amino acids [22, 29]. Here, we firstly suggested that the NtLHT1 gene in tobacco was able to transport a wide spectrum of amino acids, including acidic, basic and neutral amino acids (Fig. 4), which agreed with the previous works. In addition, the transgene-free ntlht1 mutants with higher amino acids contents in mature leaves would be well used as the valuable materials in tobacco breeding, and the question why the most amino acids contents of ntlht1 mutants less at rosette stage and flower-bud appearing stage, higher at maturity stage than those in the wild type remains to be addressed in subsequent studies.

In summary, we provide an improved effective CRISPR/Cas9 system, pOREU3TR, for tobacco genome editing using an OsU3-tRNA promoter combination to drive sgRNA expression that generates improved mutagenesis efficiency over the existing one, and a visible marker Ros1 expression cassette to monitor the transgenic events that making the screening procedure simple and efficient. The efficacy of such system was verified by the NtLHT1 gene. Thus, the improvements should result in important savings for research groups using CRISPR/Cas9 in tobacco.

Plant Materials and Growth Conditions

Nicotiana tabacum L. variety ‘Hong Hua Da Jin Yuan’ (hereinafter referred to as HD) plants were grown at 50% − 60% relative humidity and 25°C 16 h/8 h light/dark cycle in a chamber for 3 weeks before leaves were used for Agrobacterium transformation [30].

Vector Construction

All the primers used for in this study were provided in Supplementary Table S1. Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pORE-Cas9, which contains the CRISPR/Cas9 system, was kindly provided by Prof. Qingyou Xia (Southwestern University, Chongqing, China).

To inspect the efficacy of the OsU3-tRNA, AtU6-tRNA and AtU6 promoters in driving the expression of sgRNA, the OsU3-tRNA-sgRNA-T_polyT, and AtU6-tRNA-sgRNA-T_polyT sequences were synthesized by Nanjing GenScript Co (GenScript, Nanjing), and cloned into the pORE-Cas9 vector instead of the AtU6-sgRNA-T_polyT via the HindIII and SbfI sites through homologous recombination (HR) using the In-Fusion HD cloning kit (Takara), getting the two new vectors OsU3-tRNA and AtU6-tRNA, respectively. Then the tobacco PDS sgRNA was inserted into the BsaI site of the 3 vectors by restriction digestion and ligation method, obtaining the vectors of OsU3-tRNA-/AtU6-tRNA-/AtU6-PDS, respectively (Fig. 1a). The correct clones were confirmed by sequencing with primer RB-F. Similarly, to further optimize the vector, the tobacco NtLHT1 was ligated at the BsaI site in the OsU3-tRNA vector and the AtUb10-Ros1-T_NOS expression cassette was synthesized (GenScript) and fused into OsU3-tRNA-LHT1 vector at NotI and KpnI sites by HR. The improved vector was named pOREU3TR-NtLHT1 and the correct clones were identified with EXT-F primer (Fig. 2).

Plant Transformation

The CRISPR vectors were introduced into the Agrobacterium strain LBA4404, and Agrobacterium-mediated transformation of HD leaves was performed by following the protocol of leaf disc transformation [31, 32]. The tobacco calluses were subcultured on the MS3 medium plates (4.4 g l^− 1 MS medium including vitamins, 30 g l^− 1 sucrose, 2 mg l^− 1 benzylaminopurine, 0.5mg l^− 1 Naphthaleneacetic acid, 250 mg l^− 1 carbenicillin, 50 mg l^− 1 kanamycin and 4 g l^− 1 phytagel, pH 5.8) every 2 weeks until the development of kanamycin-resistant calluses. The resistant calluses were sampled for gDNA isolation, and Cas9-1F/R, PDS-1F/R primers (Supplementary Table S1) were used to identify T-DNA insertions and the editing forms of target fragments by PCR amplification and Sanger sequencing. After culture for 6–8 weeks, the induced regenerated shoots were excised and transferred to the rooting medium (4.4 g l^− 1 MS medium including vitamins, 30 g l^− 1 sucrose, 250 mg l^− 1 carbenicillin, 100 mg l^− 1 kanamycin and 4 g l^− 1 phytagel, pH 5.8), and grown for 2–3 weeks to a height of 5–6 cm before transfer to soil. The regenerated plants were sampled for gDNA extraction and molecular detection. For the pOREU3TR-NtLHT1 vector, the T₀ plants were visually screened for color phenotype at calli culture stage and at different growing stages. Seeds from each individual T₀ plants containing dark red color in stems and leaves were harvested separately. Cas9-1F/R and LHT1-1F/R primers (Supplementary Table S1) were used for the detection of transgenes and editing forms.

Qrt-pcr Assay

Total RNA of HD and T₀ plants of OsU3-tRNA-/AtU6-tRNA-/AtU6-PDS were extracted using Eastep Super Total RNA Isolation Kit (Promega, Shanghai, China), and cDNA was synthesized using the HiScript II Q RT SuperMix for qPCR Kit (Vazyme) following the manufacturer’s protocols. qRT-PCR assays were performed to determine the expression levels of the sgRNA in tobacco leaves, and tobacco NtEF1 (Ntab0421890) was used as internal control. The Primers used for qRT-PCR were described in Supplementary Table S1. The relative expression levels were calculated using the 2^−ΔΔCTmethod.

Detection Of T Plants

We randomly selected the T₁ progenies of 3 homozygous mutant T₀ plants of pOREU3TR-NtLHT1 to determine the efficiency of the new CRISPR/Cas9 system in editing the target genes. Cas9-1F/R was used to detect the presence of the T-DNA, and LHT1-1F/R was used to amplify part of the NtLHT1 gene from the normal green T₁ plants. We also obtained the seeds separately from the transgene-free and gene-edited T₁ pants.

Characterization Of T Plants

The wild type HD and T₂ plants of pOREU3TR-NtLHT1 were planted in soil in the greenhouse. We directly measured the plant height of the plants at rosette stage, flower-bud appearing stage and mature stage, and the tobacco leaves of these stages were sampled to determine the amino acid contents by HPLC (Agilent 1100, USA) [33].

Acknowledgements

The author would like to thank Prof. Qingyou Xia for kindly providing the Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pORE-Cas9, which contains the CRISPR/Cas9 system.

Author contributions

JZ carried out the experiments and data analysis. JZ and JX wrote the main manuscript text. JZ, HX, QG and XL designed and supervised the research. QM, WY, HX, LX and WZ obtained and labeled the pictures. JW, LD, JJ and GY revised the manuscript. All the authors read and approved the final manuscript.

Funding

This work was financially supported by the Research Foundation of China Tobacco Company (110202101034 (JY-11)) and the Research Foundation of China Tobacco Yunnan Industrial Co., Ltd. (2021JC08).

Availability of data and materials

All data and material generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests

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Highly efficient transgene-free plant genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR

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Abstract

Figures

Background

Results

OsU3-tRNA promoter combination enhanced the editing efficiency

Ros1 Improved The Screening Efficiency Of The Transgene-free And Gene-edited Plants

Discussion

Conclusion

Methods

Plant Materials and Growth Conditions

Vector Construction

Plant Transformation

Qrt-pcr Assay

Detection Of T Plants

Characterization Of T Plants

Declarations

References

Additional Declarations

Supplementary Files

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