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ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 2  |  Page : 44-47

Mutagenesis of cellulose synthase (CesA-Like) Gene in tomato using clustered regularly interspaced short palindromic repeat/CAS9-system


Department of Plant Science, Kulliyyah of Science, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia

Date of Submission30-Dec-2019
Date of Acceptance09-Jan-2020
Date of Web Publication23-Mar-2020

Correspondence Address:
Nurul Hidayah Samsulrizal
International Islamic University Malaysia
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/MTSP.MTSP_11_19

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  Abstract 


Background: During ripening, the changes in texture involved remodelling of cell walls of fruits including tomato and also alterations in tissue water relations caused by modifications in the cuticle. Aims and Objectives: To better understand the relationship between cell wall remodelling and fruit softening, an understanding of cell wall structure is necessary. Materials and Methods: Cellulose synthase gene that consists of cellulose synthase (CesA-like) plays important role in cellulose biosynthesis. However, CesA family genes are yet to be fully characterized in Solanaceae species. Results: In this study, we generated transgenic plants to test the role of CesA like gene in texture changes using tomato (Solanum lycopersicum) as a model system. We used the recently developed clustered regularly interspaced short palindromic repeat/Cas9 DNA editing technology to generate mutations in the target gene. Conclusion: Nevertheless, there was no mutation recovered in the CesA-like gene, and this indicates that this gene product is likely essential for regeneration of plantlets from tissue culture.

Keywords: Cell wall, cellulose synthase, clustered regularly interspaced short palindromic repeat-Cas09, gene editing, tomato


How to cite this article:
Samsulrizal NH. Mutagenesis of cellulose synthase (CesA-Like) Gene in tomato using clustered regularly interspaced short palindromic repeat/CAS9-system. Matrix Sci Pharma 2019;3:44-7

How to cite this URL:
Samsulrizal NH. Mutagenesis of cellulose synthase (CesA-Like) Gene in tomato using clustered regularly interspaced short palindromic repeat/CAS9-system. Matrix Sci Pharma [serial online] 2019 [cited 2020 May 29];3:44-7. Available from: http://www.matrixscipharma.org/text.asp?2019/3/2/44/281231




  Introduction Top


The development of methods for genome editing has progressed rapidly in the last two decades. Several methods have been developed including the use of zinc finger nucleases[1] and transcription activator-like effector nucleases.[2] More recently, the clustered regularly interspaced short palindromic repeat (CRISPR) associated-Cas9 endonuclease has been used.[3]

According to Feng et al.,[4] CRISPR/Cas9 system successfully generated mutation using Cas9 driven from CaMV 35S promoter and the synthetic sgRNA from the AtU6-26 promoter in Arabidopsis or OsU6-2 promoter in rice. A customized sgRNA encoded by a sequence of ~100 nt is required to target a specific sequence. Cas9 does not have to be reengineered for each new target site. Thus, the sgRNA: Cas9 system is therefore much more straightforward than RNA interference. Thefirst report for the use of CRISPR/Cas9 in tomato was by Ron et al.[5] and Brooks et al.[6] Ito et al.[7] also reported the efficiency of CRISPR/Cas9 method to silence the RIN gene where the mutations that contain insertion or deletion resulted in inhibition of fruit ripening.

Hence, the aims of this study are to investigate the function of the CesA-like gene in the transgenic tomato lines by generating a knockout using CRISPR/Cas9 DNA editing and examine the influence of this mutation on tomato fruit softening. In plants, most of cellulose synthase or CesA-like gene is involved during cellulose and hemicellulose biosynthesis. During fruit development, cellulose synthases are highly expressed in tomato and then decreased at the breaker stage.[8] They are likely to be involved in the biosynthesis of cellulose,[9] but till now not much studies regarding this gene is done in tomato (Solanum lycopersicum).[10]


  Materials and Methods Top


Plant materials

Tomato (S. lycopersicum) plants cv. Ailsa Craig, wild type and those containing CRISPR/Cas9 constructs were grown under standard glasshouse conditions with 16 h of day length and temperatures of 20°C during the daytime and 16°C at night. Supplemental lighting was supplied when required.

Vector construction for generating stable transgenic plants

Primers were designed [Table 1] using CesA-like sequence (Solyc08 g061100) from tomato genome browser at www.solgenomics.net. A specific sequence within the gene was then chosen to represent the guide RNA [Figure 1]. To generate the stable transgenic plants, constructs Level 1 (pICH47751AtU6p sgRNA, pICH47732NOSpNPTII-OCST, pIcH47742:35Sp Cas9-NOST, Pich41766 Linker) were used and assembled to Level 2 (pAGM4723) through the Golden gate cloning method.[11]
Table 1: Primer sequences of gene for cellulose synthase

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Figure 1: Cas9/sgRNA-mediated mutagenesis in Solanum lycopersicum. Scheme for Cas9/sgRNA-mediated mutagenesis of a nonfunctional (out-of-frame) mutant of Solyc08g061100 coding sequence

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Generation of stable transgenic plants

The final Level 2 Cas9/sgRNA construct was transformed into Agrobacterium EHA105 by electroporation. Method described by Smith et al.[12] was used to generate stable transgenic plant in growth room at 25°C with 16/8 h:light/dark photoperiod.

Identification of the transgenic lines

To identify the presence of T-DNA in the transgenic plant lines, a set of polymerase chain reaction (PCR) reactions was performed using CesA-like gene-specific primer and the NPTIICas9 primer. The DNA from wild-type Ailsa Craig was used as a positive control for the gene-specific primer pair and as negative control for T-DNA mutation. The sequence of transgene was confirmed by sequencing.

Identification of single copy homozygous lines

The T0 lines were grown to fruiting and T1 seeds were collected. Then, 10–12 T1 seeds were then sown for each of the line. The DNA from each of the plants was extracted using DNeasy Plant Mini Kit (Qiagen). PCR was used to establish the presence of the Cas9 gene and also to obtain the edited CesA-like sequences. The PCR products were then sent for sequencing.


  Results and Discussion Top


Vector construction of PSY1

According to the product sizes, most convenient primer products is 8CeSa061100 where the PCR amplicons of the target genes with the length as ~ 100 bp. This product was then cloned into vector pICSL01009AtU6p (SpecR) and shown to be correct by sequencing. By using Golden Gate cloning approach, Level 1 constructs were assembled to Level 2 vector that involved pICH47751AtU6psgRNA, pICH47732:NOSp: NPTII-OCST, pIcH4774235Sp Cas9-NOST, Pich41766 Linker and pAGM4723. The presence of DNA for CESA-like in Level 2 construct was confirmed using PCR. The destination vector Level 2 Cas9/sgRNA construct was then transformed into Agrobacterium EHA105 by electroporation. [Figure 2] shows the PCR products that were amplified from the isolated plasmid DNA (Level 2) for samples CesA-like using NPTII/Cas9-specific primer.
Figure 2: Polymerase chain reaction products were amplified using the Cas9/NPTII-specific primers from Agrobacterium EHA105. The purified polymerase chain reaction products were separated by electrophoresis on a 2% (w/v) agarose gel in 0.5X TAE at 100 V for 30 minutes. Lane 1, 2 and 3 are polymerase chain reaction products of sgRNA constructs which were isolated from colonies of Agrobacterium EHA105, Lane 4: Hyperladder marker

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Analysis of the CesA-like transgenic lines

There were seven transgenic plant lines regenerated using the CesA-like construct. These transgenic plants were then transferred to the glasshouse. To confirm the presence of the transgenes, DNA was extracted from leaves of the putative transformants. Primers NPTII/Cas9 was used to amplify the Cas9 region where the size of the product is 1600 bp [Figure 3]. The PCR generated an amplicon of the correct size of 1600 bp in putatively transformed plant lines No. 1, 2, 3, 4, 5, 6 and 7 for CesA-like. In addition, no PCR amplification product was recovered in WT plants. The experiment demonstrated that these lines contained the Cas9 gene, but it does not indicate if the endogenous gene has been mutated.
Figure 3: Polymerase chain reaction genotyping of seven representatives (a) CRISPR::CesA-like plants showing the transgenic lines that contain Cas9/ sgRNA gene construct in T0 plants

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Validation of clustered regularly interspaced short palindromic repeat/Cas9 driven mutations in the CesA-like

A PCR coupled with restriction digestion strategy as well as sequencing was used to validate the presence of mutations in the target genes of T0 lines [Figure 4]a. A PCR approach for the CesA-like gene was designed to give a single amplicon of 300 bp containing a BssSI site [Figure 4]b. The guide RNA would be expected to target sequences in the region of this restriction site. This would result modification of sequence at the site and prevent subsequent digestion by BssSI.
Figure 4: Efficiency of Cas9/sgRNA mutagenesis. (a) Polymerase chain reaction amplification of the clustered regularly interspaced short palindromic repeat: CesA-like lines to generate a 300 bp product that could then be challenged with BssS1. (b) Polymerase chain reaction/ restriction digestion analysis demonstrates that all products can be cut with BssS1 indicating a lack of mutations in this sequence

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However, the BssSI digestion pattern of PCR-amplified DNA from the 7 individual T0 plants shows similar patterns to wild type. All of the amplification products were digested by the enzyme. This suggests that the CRISPR/Cas9 targeting of the CesA-like gene has not been successful. Thus, to confirm this analysis, the PCR products from the seven T0 were purified and sent for sequencing. The sequencing results confirmed the absence of mutations in the target site of CesA-like gene [Figure 5]. One explanation for these results is that the CesA-like gene is essential for proper regeneration of plants in tissue culture, and knocking out this gene is lethal.
Figure 5: Confirmation of inheritance of a modified or nonmodified CesA-like gene. Sequence alignment and DNA sequencing traces from sequencing of polymerase chain reaction amplification of clustered regularly interspaced short palindromic repeat::CesA-like as target sites where there are no modification in the sequence level

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  Conclusion Top


The failure to regenerate CRISPR/Cas9-driven mutations in the CesA-like gene suggests that the gene is necessary for plant development. It also suggests that in some instances, CRISPR will not be the technology of choice for transgenic experiments where complete silencing of a target gene can be lethal.

Acknowledgments

I like to acknowledge University of Nottingham, UK for providing the skills and facilities. I also like to thank Jones, V. Nekrasov, S. Kamoun, T. S. L. and The Gatsby Charitable Foundation for providing facilities and provision of the CRISPR/Cas 9 vectors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 1996;93:1156-60.  Back to cited text no. 1
    
2.
Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 2010;186:757-61.  Back to cited text no. 2
    
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Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337:816-21.  Back to cited text no. 3
    
4.
Feng Z, Zhang B, Ding W, Liu X, Yang DL, Wei P, et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 2013;23:1229-32.  Back to cited text no. 4
    
5.
Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K, et al. Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 2014;166:455-69.  Back to cited text no. 5
    
6.
Brooks C, Nekrasov V, Lippman ZB, Van Eck J. Efficient gene editing in tomato in thefirst generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 2014;166:1292-7.  Back to cited text no. 6
    
7.
Ito Y, Nishizawa-Yokoi A, Endo M, Mikami M, Toki S. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening. Biochem Biophys Res Commun 2015;467:76-82.  Back to cited text no. 7
    
8.
Tomato Genome Consortium. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 2012;485:635-41.  Back to cited text no. 8
    
9.
Delmer DP, Haigler CH. The regulation of metabolic flux to cellulose, a major sink for carbon in plants. Metab Eng 2002;4:22-8.  Back to cited text no. 9
    
10.
Song X, Xu L, Yu J, Tian P, Hu X, Wang Q, et al. Genome-wide characterization of the cellulose synthase gene superfamily in Solanum lycopersicum. Gene 2019;688:71-83.  Back to cited text no. 10
    
11.
Weber E, Gruetzner R, Werner S, Engler C, Marillonnet S. Assembly of designer TAL effectors by Golden Gate cloning. PLoS One 2011;6:e19722.  Back to cited text no. 11
    
12.
Smith CJ, Watson CF, Morris PC, Bird CR, Seymour GB, Gray JE, et al. Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol Biol 1990;14:369-79.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
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