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CRISPR/Cas9-mediated gene-editing technology in fruit quality improvement

CRISPR/Cas9-mediated gene-editing technology in fruit quality improvement Fruits are an essential part of a healthy, balanced diet and it is particularly important for fibre, essential vitamins, and trace elements. Improvement in the quality of fruit and elongation of shelf life are crucial goals for researchers. However, traditional techniques have some drawbacks, such as long period, low efficiency, and difficulty in the modification of target genes, which limit the progress of the study. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique was developed and has become the most popular gene-editing technology with high efficiency, simplicity, and low cost. CRISPR/Cas9 technique is widely accepted to analyse gene function and complete genetic modification. This review introduces the latest progress of CRISPR/Cas9 technology in fruit quality improvement. For example, CRISPR/Cas9-mediated targeted mutagenesis of RIPENING INHIBITOR gene (RIN), Lycopene desaturase (PDS), Pectate lyases (PL), SlMYB12, and CLAVATA3 (CLV3) can affect fruit ripening, fruit bioactive compounds, fruit texture, fruit colouration, and fruit size. CRISPR/Cas9-mediated mutagenesis has become an efficient method to modify target genes and improve fruit quality. Key words: fruit; CRISPR/Cas9; fruit quality. biotechnology to modify target genes, has been developed rapidly Introduction and efficiently. At present, the genome sequences of many species, including Gene editing technology mainly includes zinc-finger nucleases model plants, crops, and medicinal species, have been completed. (ZFNs; Takatsuji, 1999), transcription activator-like effector nu- Researchers have been focussing on the study of gene function, cleases (TALENs; Li et  al., 2011), and clustered regularly inter- genetic modification, and genetic improvement for decades. Gene spaced short palindromic repeats (CRISPR; Barrangou et al., 2007; modification technology can carry out site-specific knockout, re- Ran et  al., 2013; Mao et  al., 2019). These technologies usually in- placement, mutation, and introduction of exogenous genes in volve the application of sequence-specific nucleases to identify the genome. There are three main types of gene modification tech- sequences connected to the nuclease domain, which can precisely nologies including gene targeting, RNA interference (RNAi), and target double-strand DNA to produce double-strand break (DSB). engineered endonuclease (Zhou et al., 2019). These three kinds of DSB prompts cells to initiate two major DNA damage repair techniques have been widely used in gene modification. However, mechanisms: non-homologous end joining (NHEJ) and homology- the techniques have some drawbacks, such as long transform- ation period, low efficiency, and difficulty in the modification of directed repair (HDR; Moore and Haber, 1996; Haber et al., 2004). the target gene. Recently, gene-editing technology, as emerging In the NHEJ repair, the broken ends of the double strands can be © The Author(s) 2020. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/fqs/article/4/4/159/5940658 by DeepDyve user on 22 December 2020 160 X. Xu et al., 2020, Vol. 4, No. 4 directly pulled closer to each other by repairing proteins and re- 2016). Transgenic technology is also used for the regulation of gene joined with the help of DNA ligase (Budman and Chu, 2005). HDR expression, which may have the characteristics of a long cycle, low is a mechanism to repair DNA double-strand damage in cells and safety, and cumbersome process (Martin and Caplen, 2007). Virus- can only occur when there are DNA fragments homologous to the induced gene silencing (VIGS) technology has been widely used in the damaged DNA in the nucleus (Zha et  al., 2009). Therefore, HDR field for plant gene function research because of its simplicity, high ef- can introduce a specific point mutation sequence by providing ex- ficiency, and no need to rely on transgenic operations. However, com- ogenous donor template. The repair processes of NHEJ and HDR pared with transgenic technology, VIGS is of low cost and fast and are shown in Figure 1. ZFNs are the first generation of gene editing many viral silencing vectors only induce a short-term silent phenotype nuclease, which is formed by the fusion of transcription factors con- (Ding et al., 2018). In addition, many genetic engineering technolo- taining the zinc finger domain and the cutting domain of FokI endo- gies have emerged that can accurately modify specific target genes in nuclease (Mahfouz et  al., 2011). FokI protein, a type IIS nuclease, the genome of organisms. Yang et  al. (2017) used CRISPR/Cas9 to contains an N-terminal DNA-binding domain and a non-specific generate SlORRM4 gene mutants and obtained slorrm4 mutant lines C-terminal DNA-cleavage domain (Kim et  al., 1997). ZFNs tech- with delayed fruit ripening. In 2018, CRISPR/Cas9-mediated muta- nology is a DNA-targeted modification technology, which is widely tions of long non-coding RNA1459 (lncRNA1459) locus resulted in used in genome targeted modification. But the complex construction an inhibition of fruit ripening (Li et  al., 2018b). Gene-editing tech- process, high cost, high off-target rate, and high toxicity to cells limit nology is an indispensable tool for the study of gene functions and the the development of ZFNs technique (Gaj et al., 2013). TALENs are genetic improvement of fruit crops. nucleic acid endonucleases formed by the fusion of specific DNA- binding domain and non-specific endonuclease FokI cleavage do- CRISPR/Cas9 Gene Editing Technique main, which recognizes the correspondence between single protein and nucleotide (Joung and Sander, 2013). Compared with the ZFNs, The CRISPR/Cas9 system consists of CRISPR sequence and Cas9 TALENs technique is easier to assemble and design and has a lower protein. CRISPR sequence is composed of some highly conserva- off-target effect (Mahfouz et al., 2011). CRISPR/Cas9 was first dis- tive repetitive sequence and interval sequence and Cas9 protein en- covered in 1987 in the flanks of the iap gene sequence of K12 in coded by related genes near the CRISPR sequence. Cas9 protein has Escherichia coli (Ishino et  al., 1987). In recent decades, CRISPR/ nuclease activity and can cut the DNA sequence, leading to DNA Cas9 technology has developed to become the most popular gene- DSB (Barrangou, 2013). The earliest known CRISPR/Cas9 system editing technology. CRISPR/Cas9 technology is a new technology is an autoimmune defense mechanism that can resist foreign DNA that uses specific nucleases to edit the genome under the guidance of invasion (bacteriophages, plasmids, etc.). When exogenous DNA specific RNA (Sampson and Weiss, 2014). Compared with the ZFNs invades, the process of DNA signalling to RNA is disrupted, and and TALENs, CRISPR/Cas9 technique can achieve fixed-point modi- CRISPR RNA (crRNA), trans-activated RNA (tracer-RNA), and fication of DNA, which is easy to customize and highly efficient in Cas9 nucleases work together to destroy the binding sites of the target shooting (Makarova et al., 2015). Therefore, many researchers invading DNA, thus protecting the host bacteria (Zhou et al., 2020). choose CRISPR/Cas9 gene-editing tool to conduct a qualitative and In general, the CRISPR/Cas9 gene editing mainly consists of three locational analysis of one or more genes (Puchta, 2017). processes: adaptation, obtaining new spacers from invading elem- Fruits contain fibre, vitamins, minerals, and bioactive com- ents and transferring spacers into the CRISPR site for immunity; pounds, which are major nutrients for human health and prevent the production of crRNA, in which the CRISPR locus is transcribed occurrence of many diseases (Giovannoni, 2007; Singh et al., 2016; and processed into small interfering crRNA; interference, in which Wang et al., 2019d). Fruit can also be used as a staple food. Bananas crRNA directs the Cas9 mechanism to specifically clear invasive nu- and plantains are used as the main food in some tropical regions cleic acids (Barrangou, 2013). The working principle of the CRISPR/ (Giovannoni et  al., 2017). Fruit crops have been threatened by the Cas9 of Streptococcus pyogenes type II was demonstrated by Jinek external environment and other factors, such as drought, cold, and et al. (2012) and it was proposed that Cas9 could cleave the double disease. Some fruit crops have a low fruit setting rate and are easy strand of target DNA under the guidance of recombinant small RNA to rot. In order to improve fruit quality and stabilize the supply of molecule (sgRNA; Martin et al., 2012; Figure 2). fruit, the crops were domesticated from wild plants from generation Hwang et  al. (2013) reported that synthetic sgRNAs could to generation or selected by the sexual hybridization of suitable guide Cas9 endogenous nucleases to modify zebrafish embryo genes parents (Hickey et al., 2017). However, this screening method has a (Hwang et  al., 2013). Since 2013, researchers have published sev- long cycle, large randomness, and a high mutation rate (Yusuff et al., eral articles on the CRISPR/Cas9 system in Science and Nature Biotechnology, which reported that precise genetic modification has been successfully achieved in mice, zebrafish, and other species. At the same time, the CRISPR/Cas9 system was optimized, including Figure 1. The repair processes of NHEJ and HDR.  NHEJ, non-homologous Figure 2. The mechanism of the CRISP/Cas9 system. sgRNA, small RNA end joining; HDR, homology-directed repair; DSB, double-strand break. molecule. Downloaded from https://academic.oup.com/fqs/article/4/4/159/5940658 by DeepDyve user on 22 December 2020 Gene Editing Technology in Fruit Quality Improvement, 2020, Vol. 4, No. 4 161 the optimization of Cas protein, promoter, and sgRNA. Now, in add- phenotypes of RNAi silencing lines and spontaneous mutants. Wang ition to Cas9, Cas12a (Cpf1), Cas13a (C2c2), nCas9, and dCas9 et  al. (2019c) obtained knockout mutants of AP2a, FUL1, FUL2, are also used in the optimized CRISPR/Cas9 system for the study and NOR genes in tomato by CRISPR/cas9 technology. The nor of microbial immunity, nucleic acid detection, and plant defense spontaneous mutant fruits show green, but the nor mutant produced mechanisms (Fauser et  al., 2014; Zetsche et  al., 2015; Gootenberg by CRISPR/Cas9 exhibits earlier ripening and orange ripe pheno- et al., 2017). The optimized YAO, SPL, DMC1, and MGE promoters types. Compared with spontaneous nor mutants, nor mutants have are used in studying the cell division and crop improvement of a milder phenotype (Wang et  al., 2020). The ful1 or ful2 double Arabidopsis, citrus, and maize (Mao et al., 2016; Zhang et al., 2017; mutant shows a severe blocked ripening phenotype (Marian et  al., Feng et al., 2018; Xu et al., 2018). Multiple sgRNA expression cas- 2014; Wang et  al., 2019b). However, the ful1 and ful2 single mu- settes are constructed into one CRISPR/Cas9 vector, and a single tants exhibit normal ripening phenotypes, indicating that FUL1 and polycistron gene is used to produce a large number of sgRNA, which FUL2 have a redundant function in the fruit ripening process. is used to study the gene function in maize and tobacco (Gao et al., The NOR and colourless non-ripening (CNR) genes are knocked 2015; Char et al., 2017). CRISPR/Cas9 system has been applied to out using CRISPR/Cas9-mediated mutagenesis and the mutants the study of fruit ripening and quality. showed only delayed or partial immature phenotypes, which is dif- ferent from their spontaneous mutants (Gao et  al., 2019). CRISPR/ Cas9 technology has become the main method to re-evaluate the Application of Gene Editing in Fruit Ripening important gene in fruit ripening through the generation of null mu- tant. The tomato nor mutant produces a new protein, 186-amino- The fleshy fruit undergoes a developmental process and ends with an acid protein (NOR186), which has a prohibitive function that affects irreversible maturation process. A lot of physiological, biochemical, ripening. The tomato nor mutant has previously been proven to be a and structural changes have taken place during the ripening process gain-of-function mutant, but the specific mechanism of action is still of fruit, as a result, it can attract more seed spreaders (Gapper et al., unclear (Wang et  al., 2019a). In nor natural mutants, the NOR186 2013). After the fruit reaches the optimum edible stage, the fruit will protein can still enter the nucleus, which can bind but cannot acti- slowly deteriorate and the fruit quality will be reduced. Therefore, vate the promoters of the key genes SlACS2, SlGgpps2, and SlPL for the regulation of fruit ripening has become the focus of many scien- ethylene biosynthesis, carotenoid accumulation, and fruit softening. tists (Martín-Pizarro and Posé, 2018). The activation effect of NOR protein on the above-mentioned pro- RIPENING INHIBITOR gene (RIN) belongs to the MADS-box moter will be inhibited by NOR186. The above research results fur- gene family and mutations of this gene can inhibit the ripening of ther prove that the nor natural mutant is a gain-of-function mutant, tomato fruits (Vrebalov et al., 2002). RIN is thought to be the main and the truncated protein NOR186 produced by the mutation of the regulatory gene in the maturation of tomato fruits and activates NOR gene has a dominant negative function (Gao et al., 2020). A re- and promotes all physiological processes associated with ripening, cent study indicated that VIGS technology can mediate nor-like1 silen- including colour, hardness, and flavour (Fujisawa et  al., 2013). The cing, which can suppress tomato fruit ripening. The inactive mutant of RIN gene is knocked out in tomato by CRISPR/Cas9 system and the nor-like1 was obtained through CRISPR/Cas9 technology, which de- rin mutant is analysed (Ito et al., 2015). The rin knockout mutation layed fruit ripening and inhibited ethylene, carotenoid synthesis, and does not affect the initiation of ripening and exhibits moderate red fruit softening. The above results indicate that nor-like1 gene plays a colouring, which demonstrates that RIN gene is not required for the positive regulatory role in tomato fruit ripening (Gao et al., 2018). ripening initiation in fruit (Ito et al., 2017). The rin mutant can lead According to reports, epigenetic modification and fruit ripening to ripening failure, which is caused by the deletion part of the DNA are inseparable. It has been found that the early epigenetic mark fragment between rin and the adjacent gene MACROCALYX (MC) is DNA cytosine methylation in the plant genome, which respond (Vrebalov et al., 2002). Due to the partial deletion of DNA fragments, to external environmental stress, regulate gene expression, and sta- the transcription factor MADS-RIN and MADS-MC are fused to bilize the genome (Chen et  al., 2018b). SlDML2 is closely related form the function of the fusion protein RIN–MC. The RIN–MC fu- to Arabidopsis DNA demethylase gene ROS1. SlDML2 knockout sion protein encodes a new transcription factor, which regulates the mutants were obtained by the CRISPR/Cas9 system, which inhibits expression of downstream genes and inhibits fruit ripening, which fruit ripening (Zhou et al., 2019). has a negative regulatory effect. (Vrebalov et  al., 2002; Ito et  al., In summary, compared with traditional knockdown technology, 2017; Li et  al., 2018d). Osorio et  al. (2020) reported that tomato mutants obtained by CRISPR/Cas technology can lead to unex- breeders use RIN mutation to acquire improved hybrids which exert pected weak phenotypes, which indicate that the compensation a negative impact on tomato flavour. Li et al. (2020) pointed out that mechanism in the body may obscure protein function. This requires RIN-deficient fruits obtained by CRISPR/Cas9 technology can reduce us to re-evaluate the model of the regulation of ripening, which may ethylene production and affect the synthesis of volatile substances involve a complex network of redundant components (Wang et al., and carotenoids. The low ethylene production is due to the fact that 2020). This reminds us to carefully design experimental programs RIN-deficient fruits cannot induce the production of ethylene in the when using CRISPR/Cas technology to evaluate other mechanisms. autocatalytic system-2. They also lack volatiles and carotenoids and transcripts related to these pathways. Meanwhile, Li et  al. (2020) supported that the fruit ripening process requires the participation of Gene Editing in Fruit Bioactive Compounds ERFs, RIN, and ethylene. Ethylene initiates the maturation of green fruit and affects the expression of RIN and other factors, which com- Many natural biologically active substances in fresh fruits have plete the whole ripening process of fruits (Li et al., 2020). anti-inflammatory, anti-cancer, anti-oxidation, and other physiological APETALA2a (AP2a), NON-RIPENING (NOR), and activities. Lycopene, carotenoid, anthocyanin, and gamma-aminobu- FRUITFULL (FUL1/TDR4 and FUL2/MBP7) play important roles tyric acid (GABA) are the main functional factors in fresh fruits. in fruit ripening (Mordy et  al., 1998; Chung et  al., 2010; Bemer Therefore, enhanced accumulation of bioactive substances has been et  al., 2012). But these results were derived from analysis of the focussed on by numerous studies (Amish et al., 2015). Downloaded from https://academic.oup.com/fqs/article/4/4/159/5940658 by DeepDyve user on 22 December 2020 162 X. Xu et al., 2020, Vol. 4, No. 4 Carotenoids play a protective role in ROS-mediated disorders Pectate lyases (PL) is an important component of pectinase. It is a and photosensitive or eye-related disorders. Carotenoids are mostly depolymerase that can degrade plant cell walls and lead to the soft- C40 terpenoids, which affect the growth, development, and matur- ening and even death of plant tissues (Uluisik and Seymour, 2020). ation of plants and improve the oxidative stability of poultry prod- The mutation of tomato PL gene is induced by CRISPR/cas9, which ucts such as egg and meat (Domonkos et  al., 2013; Nisar et  al., increases the firmness of the fruit and prolongs the shelf life of the 2015; Nabi et al., 2020). fruit, without negatively affecting other aspects of fruit ripening Lycopene is an acyclic carotenoid and red pigment that is abun- (Uluisik et al., 2016). dant in many ripening fruits. Lycopene reduces the risk of a variety Many ripening spontaneous mutants such as rin, nor, crn, and alc of tumours, including prostate cancer, and cardiovascular disease (Li can prolong storage time. The alc mutant has one base pair mutation and Xu, 2014; Tang et  al., 2014). In the process of fruit ripening, of NOR gene, resulting in nonsynonymous amino acid change (Yu lycopene is decreased due to the conversion to β-carotene and et al., 2017). Compared with the rin and nor mutants, the alc muta- α-carotene. In tomato, Li et al. (2018c) knocked out SGR1, LCY-E, tion not only prolongs shelf life, but also has better flavour and better BLC, LCY-B1, and LCY-B2 by the CRISPR/Cas9 method, which disease resistance. Tomato ALC gene mutation is obtained by using inhibited the conversion of lycopene and increased the lycopene the CRISPR/Cas9 method through HDR recombination pathway. content in the fruit about 5.1 times. During tomato fruit ripening, The alc homozygous mutant without T-DNA insertion exhibits im- phytoene synthase 1 (PSY1) is involved in the formation of lycopene proved storage time and prolonged shelf life (Yu et al., 2017). (Fray and Grierson,1993; Giorio et al., 2008). Gene editing of PSY1 was performed using CRISPR/Cas9 and the impaired PSY1 gene re- Application of Gene Editing in Fruit sults in the void of lycopene and yellow fruit, like yellow flesh mu- Colouration tant (D’Ambrosio et al., 2018). Lycopene desaturase (PDS) is an essential enzyme for the accumu- The difference in fruit colour is caused by the change of pigment. lation of lycopene and carotenoids (Bai et  al., 2016). The successful Genes affecting pigment synthesis can not only affect the bioactive mutations PDS1 and PDS2 of banana cv. Rasthali are generated by compounds, but also affect the colour of the fruit. In fruit and vege- the CRISPR/Cas9. Gene editing of the two genes resulted in the pre- table crops, colour is affecting consumer choice. For example, con- mature termination of PDS1 and PDS2 protein synthesis by inserting a sumers in Europe and the United States prefer red tomatoes, while termination codon into the gene sequences. The mutants exhibited de- Asians prefer pink tomatoes (Lin et al., 2014). The study of SlMYB12 creased chlorophyll and total carotenoid contents (Kaur et al., 2018). has proven to affect the accumulation of flavonoids. The mutation GABA, as a neuro-suppressant, has the functions of anti-fatigue, of SlMYB12 can produce pink tomato fruits (Ballester et al., 2010). sedation, and blood pressure regulation (Bachtiar et  al., 2015; The ant1 mutation is obtained by the CRISPR/cas9 system that can Takayama and Ezura, 2015). Research has proved that glutamate enhance the accumulation of anthocyanins and produce purple to- decarboxylase (GAD) catalysed the decarboxylation of glutamate to mato fruits (Čermák et al., 2015). PL, polygalacturonase 2a (PG2a), produce GABA (Akihiro et  al., 2008). GAD has a C-terminal self- and β-galactanase (TBG4) are tomato pectin degrading enzymes inhibiting region, and deletion of the domain promotes GAD activity that affect fruit ripening. Wang et  al. (2019a) obtained silent mu- (Takayama et al., 2015). In order to increase the content of GABA, tants of pl, pg2a, and tbg4. Interestingly, pg2a and tbg4 CRISPR Nonaka et  al. (2017) used the CRISPR/Cas9 method to delete the strains did not soften but affected the colour of the fruits (Wang C-terminal self-inhibitory domains of SlGAD2 and SlGAD3. The et al., 2019a). accumulation of GABA in mutant fruits increased by 7–15 times, As a widely used gene-editing technology, CRISPR/cas9 has great which affects the fruit size and yield in tomato (Nonaka et al., 2017). potential in the research of fruit colouration. Delila (Del) encodes a Previous studies have demonstrated that GABA transaminase transcription factor that has a basic helix-loop-helix (bHLH) domain (GABA-TP1, TP2, TP3), succinate semialdehyde dehydrogenase and Rosea1 (Ros1) encodes an MYB-related transcription factor (SSADH), and CAT9 are involved in GABA metabolism (Bao et al., (Goodrich et al., 1992; Schwinn et al., 2006). Butelli et al. (2008) ex- 2015; Snowden et  al., 2015). Li et  al. (2018a) successfully edited pressed the Del and Ros1 genes from snapdragon in tomato, which the five genes (GABA-TP1, GABA-TP2, GABA-TP3, SSADH, and the anthocyanin content of tomato fruit was significantly higher CAT9) in tomato genome by using pYLCRISPR/Cas9 vector, which than the accumulation of tomato anthocyanin previously reported. is a multi-locus gene knockout CRISPR/Cas9 system (Ma et  al., Engineering Del and Ros1 genes using CRISPR/Cas9 allows the cre- 2015). The multisite gene mutagenesis resulted in manipulated ation of novel mutants and holds great potential for studying the GABA metabolic pathways and significantly enhanced the GABA effect of transcription factors on fruit colouration. content (Li et al., 2018a). These studies show that CRISPR/Cas9 gene editing can be used Summary as an effective technique to modify bioactive compounds in fruits. The technology of gene editing is a powerful tool for functional gen- omics in improving the quality and commercial value of fruits. The Application of Gene Editing in Fruit Texture progresses on CRISPR/Cas9-mediated mutation involved in fruit Fruit texture is an indispensable factor in the study of fruit quality and ripening, fruit bioactive compounds, and fruit texture are summar- affects the commercial production of the fruit (Preeti et al., 2010). The ized in Table  1. The emergence of CRISPR/Cas9 technology pro- change of texture may lead to substantial decay of fruit in transporta- vides a new opportunity to accelerate plant molecular breeding. The tion and storage, which results in the development of typical diseases CRISPR/Cas9 technology used on fruit crops not only provided a during post-harvest storage and shelf life (Vicente et al., 2007). These shortcut to obtain high yield and good quality of fruit food, but texture changes are related to change the activity of many enzymes, also laid a solid foundation for fruit functional genomics research. which affect the structure of cell walls (Tucker et al., 2017). Therefore, An unavoidable problem with all gene-editing tools is the off-target fruit texture is closely related to fruit quality and shelf life. effect of non-specific site dissection of the genome. Off-target effects Downloaded from https://academic.oup.com/fqs/article/4/4/159/5940658 by DeepDyve user on 22 December 2020 Gene Editing Technology in Fruit Quality Improvement, 2020, Vol. 4, No. 4 163 Table 1. List of research on fruit improvement by using CRISPR/Cas gene-editing technology. Species Gene Gene function or phenotype Reference Tomato ALMT9 Decrease in malate content Ye et al. (2017) MPK20 Decrease in sugar content Chen et al. (2018a) ARF7 Parthenocarpic fruit Hu et al. (2018) GGP1 Ascorbic acid Li et al. (2018a) PG2a, TBG4 Fruit colour Wang et al. (2019b) SlMYB12 Fruit colour Ballester et al. (2010) ANT1 Fruit colour Čermák et al. (2015) CYCB Lycopene synthesis Zsögön et al. (2018) TBG4 Fruit firmness Wang et al. (2019b) CNR, NOR Fruit ripening Gao et al. (2019) RIN Fruit ripening Ito et al. (2017) SlEIN2, SlERFE1, SlARF2B, SlACS4 SlGRAS8, SlACS2 Fruit ripe and development Hu et al. (2019) lncRNA1459 Fruit ripening, lycopene, Li et al. (2018b) carotenoid biosynthesis SGR1, Blc, LCY-E, LCY-B1, LCY-B2 Increased lycopene content Li et al. (2018c) SlORRM4 Fruit ripening Yang et al. (2017) L1L4 Fruit metabolism Gago et al. (2017) SlAGL6 Parthenocarpic fruit Klap et al. (2017) RIN Fruit ripening Ito et al. (2015) AP2a, FUL1, FUL2, NOR Fruit ripening Wang et al. (2019b) SlDML2 Fruit ripening Zhou et al. (2019) SGR1, LCY-E, Blc, LCY-B1, LCY-B2 Lycopene synthesis Li et al. (2018c) PSY1 Lycopene synthesis D’Ambrosio et al. (2018) SlGAD2, SlGAD3 GABA content Nonaka et al. (2017) GABA-TP1, GABA content Ma et al. (2015) GABA-TP2, GABA-TP3, SSADH, CAT9 PL Fruit firmness Uluisik et al. (2016) ALC Long shelf life Yu et al. (2017) CLV3 Fruit size Zsögön et al. (2018) ENO Fruit size Yuste-Lisbona et al. (2020) Watermelon PDS Carotenoid biosynthesis Wang et al. (2019d) Banana PDS1, PDS2 Chlorophyll, Carotenoid Kaur et al. (2018) Apple IdnDH Biosynthesis of tartaric acid Osakabe et al. (2018) Grape VvPDS Albino phenotype Nakajima et al. (2017) Groundcherry ClV1 Fruit size Lemmon et al. (2018) Kiwifruit CEN Fruit development Varkonyi-Gasic et al. (2019) RIN, RIPENING INHIBITOR gene; PDS, Lycopene desaturase; PG2a, polygalacturonase 2a; TBG4, β-galactanase; CNR, colourless non- ripening; AP2a, APETALA2a; NOR, NON-RIPENING; PSY1, phytoene synthase 1; PL, Pectate lyases; CLV3, CLAVATA3; PDS, Lycopene desaturase. disrupt the expression of functional genes, affect gene functions, Conflict of Interest and eventually produce unpredictable adverse reactions (Guilinger The authors declare no conflict of interest. et al., 2014). Schaefer et al. (2017) published a peer-reviewed paper in which the authors reported that CRISPR-Cas9 caused unexpected References off-target changes in mice (Schaefer et  al., 2017). Therefore, gene- editing technology still needs to be further optimized to avoid off- Akihiro,  T., Koike,  S., Tani,  R., et  al. (2008). Biochemical mechanism on target and increase on-target editing efficiency. It is believed that GABA accumulation during fruit development in tomato. 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CRISPR/Cas9-mediated gene-editing technology in fruit quality improvement

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Oxford University Press
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© The Author(s) 2020. Published by Oxford University Press on behalf of Zhejiang University Press.
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Abstract

Fruits are an essential part of a healthy, balanced diet and it is particularly important for fibre, essential vitamins, and trace elements. Improvement in the quality of fruit and elongation of shelf life are crucial goals for researchers. However, traditional techniques have some drawbacks, such as long period, low efficiency, and difficulty in the modification of target genes, which limit the progress of the study. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique was developed and has become the most popular gene-editing technology with high efficiency, simplicity, and low cost. CRISPR/Cas9 technique is widely accepted to analyse gene function and complete genetic modification. This review introduces the latest progress of CRISPR/Cas9 technology in fruit quality improvement. For example, CRISPR/Cas9-mediated targeted mutagenesis of RIPENING INHIBITOR gene (RIN), Lycopene desaturase (PDS), Pectate lyases (PL), SlMYB12, and CLAVATA3 (CLV3) can affect fruit ripening, fruit bioactive compounds, fruit texture, fruit colouration, and fruit size. CRISPR/Cas9-mediated mutagenesis has become an efficient method to modify target genes and improve fruit quality. Key words: fruit; CRISPR/Cas9; fruit quality. biotechnology to modify target genes, has been developed rapidly Introduction and efficiently. At present, the genome sequences of many species, including Gene editing technology mainly includes zinc-finger nucleases model plants, crops, and medicinal species, have been completed. (ZFNs; Takatsuji, 1999), transcription activator-like effector nu- Researchers have been focussing on the study of gene function, cleases (TALENs; Li et  al., 2011), and clustered regularly inter- genetic modification, and genetic improvement for decades. Gene spaced short palindromic repeats (CRISPR; Barrangou et al., 2007; modification technology can carry out site-specific knockout, re- Ran et  al., 2013; Mao et  al., 2019). These technologies usually in- placement, mutation, and introduction of exogenous genes in volve the application of sequence-specific nucleases to identify the genome. There are three main types of gene modification tech- sequences connected to the nuclease domain, which can precisely nologies including gene targeting, RNA interference (RNAi), and target double-strand DNA to produce double-strand break (DSB). engineered endonuclease (Zhou et al., 2019). These three kinds of DSB prompts cells to initiate two major DNA damage repair techniques have been widely used in gene modification. However, mechanisms: non-homologous end joining (NHEJ) and homology- the techniques have some drawbacks, such as long transform- ation period, low efficiency, and difficulty in the modification of directed repair (HDR; Moore and Haber, 1996; Haber et al., 2004). the target gene. Recently, gene-editing technology, as emerging In the NHEJ repair, the broken ends of the double strands can be © The Author(s) 2020. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/fqs/article/4/4/159/5940658 by DeepDyve user on 22 December 2020 160 X. Xu et al., 2020, Vol. 4, No. 4 directly pulled closer to each other by repairing proteins and re- 2016). Transgenic technology is also used for the regulation of gene joined with the help of DNA ligase (Budman and Chu, 2005). HDR expression, which may have the characteristics of a long cycle, low is a mechanism to repair DNA double-strand damage in cells and safety, and cumbersome process (Martin and Caplen, 2007). Virus- can only occur when there are DNA fragments homologous to the induced gene silencing (VIGS) technology has been widely used in the damaged DNA in the nucleus (Zha et  al., 2009). Therefore, HDR field for plant gene function research because of its simplicity, high ef- can introduce a specific point mutation sequence by providing ex- ficiency, and no need to rely on transgenic operations. However, com- ogenous donor template. The repair processes of NHEJ and HDR pared with transgenic technology, VIGS is of low cost and fast and are shown in Figure 1. ZFNs are the first generation of gene editing many viral silencing vectors only induce a short-term silent phenotype nuclease, which is formed by the fusion of transcription factors con- (Ding et al., 2018). In addition, many genetic engineering technolo- taining the zinc finger domain and the cutting domain of FokI endo- gies have emerged that can accurately modify specific target genes in nuclease (Mahfouz et  al., 2011). FokI protein, a type IIS nuclease, the genome of organisms. Yang et  al. (2017) used CRISPR/Cas9 to contains an N-terminal DNA-binding domain and a non-specific generate SlORRM4 gene mutants and obtained slorrm4 mutant lines C-terminal DNA-cleavage domain (Kim et  al., 1997). ZFNs tech- with delayed fruit ripening. In 2018, CRISPR/Cas9-mediated muta- nology is a DNA-targeted modification technology, which is widely tions of long non-coding RNA1459 (lncRNA1459) locus resulted in used in genome targeted modification. But the complex construction an inhibition of fruit ripening (Li et  al., 2018b). Gene-editing tech- process, high cost, high off-target rate, and high toxicity to cells limit nology is an indispensable tool for the study of gene functions and the the development of ZFNs technique (Gaj et al., 2013). TALENs are genetic improvement of fruit crops. nucleic acid endonucleases formed by the fusion of specific DNA- binding domain and non-specific endonuclease FokI cleavage do- CRISPR/Cas9 Gene Editing Technique main, which recognizes the correspondence between single protein and nucleotide (Joung and Sander, 2013). Compared with the ZFNs, The CRISPR/Cas9 system consists of CRISPR sequence and Cas9 TALENs technique is easier to assemble and design and has a lower protein. CRISPR sequence is composed of some highly conserva- off-target effect (Mahfouz et al., 2011). CRISPR/Cas9 was first dis- tive repetitive sequence and interval sequence and Cas9 protein en- covered in 1987 in the flanks of the iap gene sequence of K12 in coded by related genes near the CRISPR sequence. Cas9 protein has Escherichia coli (Ishino et  al., 1987). In recent decades, CRISPR/ nuclease activity and can cut the DNA sequence, leading to DNA Cas9 technology has developed to become the most popular gene- DSB (Barrangou, 2013). The earliest known CRISPR/Cas9 system editing technology. CRISPR/Cas9 technology is a new technology is an autoimmune defense mechanism that can resist foreign DNA that uses specific nucleases to edit the genome under the guidance of invasion (bacteriophages, plasmids, etc.). When exogenous DNA specific RNA (Sampson and Weiss, 2014). Compared with the ZFNs invades, the process of DNA signalling to RNA is disrupted, and and TALENs, CRISPR/Cas9 technique can achieve fixed-point modi- CRISPR RNA (crRNA), trans-activated RNA (tracer-RNA), and fication of DNA, which is easy to customize and highly efficient in Cas9 nucleases work together to destroy the binding sites of the target shooting (Makarova et al., 2015). Therefore, many researchers invading DNA, thus protecting the host bacteria (Zhou et al., 2020). choose CRISPR/Cas9 gene-editing tool to conduct a qualitative and In general, the CRISPR/Cas9 gene editing mainly consists of three locational analysis of one or more genes (Puchta, 2017). processes: adaptation, obtaining new spacers from invading elem- Fruits contain fibre, vitamins, minerals, and bioactive com- ents and transferring spacers into the CRISPR site for immunity; pounds, which are major nutrients for human health and prevent the production of crRNA, in which the CRISPR locus is transcribed occurrence of many diseases (Giovannoni, 2007; Singh et al., 2016; and processed into small interfering crRNA; interference, in which Wang et al., 2019d). Fruit can also be used as a staple food. Bananas crRNA directs the Cas9 mechanism to specifically clear invasive nu- and plantains are used as the main food in some tropical regions cleic acids (Barrangou, 2013). The working principle of the CRISPR/ (Giovannoni et  al., 2017). Fruit crops have been threatened by the Cas9 of Streptococcus pyogenes type II was demonstrated by Jinek external environment and other factors, such as drought, cold, and et al. (2012) and it was proposed that Cas9 could cleave the double disease. Some fruit crops have a low fruit setting rate and are easy strand of target DNA under the guidance of recombinant small RNA to rot. In order to improve fruit quality and stabilize the supply of molecule (sgRNA; Martin et al., 2012; Figure 2). fruit, the crops were domesticated from wild plants from generation Hwang et  al. (2013) reported that synthetic sgRNAs could to generation or selected by the sexual hybridization of suitable guide Cas9 endogenous nucleases to modify zebrafish embryo genes parents (Hickey et al., 2017). However, this screening method has a (Hwang et  al., 2013). Since 2013, researchers have published sev- long cycle, large randomness, and a high mutation rate (Yusuff et al., eral articles on the CRISPR/Cas9 system in Science and Nature Biotechnology, which reported that precise genetic modification has been successfully achieved in mice, zebrafish, and other species. At the same time, the CRISPR/Cas9 system was optimized, including Figure 1. The repair processes of NHEJ and HDR.  NHEJ, non-homologous Figure 2. The mechanism of the CRISP/Cas9 system. sgRNA, small RNA end joining; HDR, homology-directed repair; DSB, double-strand break. molecule. Downloaded from https://academic.oup.com/fqs/article/4/4/159/5940658 by DeepDyve user on 22 December 2020 Gene Editing Technology in Fruit Quality Improvement, 2020, Vol. 4, No. 4 161 the optimization of Cas protein, promoter, and sgRNA. Now, in add- phenotypes of RNAi silencing lines and spontaneous mutants. Wang ition to Cas9, Cas12a (Cpf1), Cas13a (C2c2), nCas9, and dCas9 et  al. (2019c) obtained knockout mutants of AP2a, FUL1, FUL2, are also used in the optimized CRISPR/Cas9 system for the study and NOR genes in tomato by CRISPR/cas9 technology. The nor of microbial immunity, nucleic acid detection, and plant defense spontaneous mutant fruits show green, but the nor mutant produced mechanisms (Fauser et  al., 2014; Zetsche et  al., 2015; Gootenberg by CRISPR/Cas9 exhibits earlier ripening and orange ripe pheno- et al., 2017). The optimized YAO, SPL, DMC1, and MGE promoters types. Compared with spontaneous nor mutants, nor mutants have are used in studying the cell division and crop improvement of a milder phenotype (Wang et  al., 2020). The ful1 or ful2 double Arabidopsis, citrus, and maize (Mao et al., 2016; Zhang et al., 2017; mutant shows a severe blocked ripening phenotype (Marian et  al., Feng et al., 2018; Xu et al., 2018). Multiple sgRNA expression cas- 2014; Wang et  al., 2019b). However, the ful1 and ful2 single mu- settes are constructed into one CRISPR/Cas9 vector, and a single tants exhibit normal ripening phenotypes, indicating that FUL1 and polycistron gene is used to produce a large number of sgRNA, which FUL2 have a redundant function in the fruit ripening process. is used to study the gene function in maize and tobacco (Gao et al., The NOR and colourless non-ripening (CNR) genes are knocked 2015; Char et al., 2017). CRISPR/Cas9 system has been applied to out using CRISPR/Cas9-mediated mutagenesis and the mutants the study of fruit ripening and quality. showed only delayed or partial immature phenotypes, which is dif- ferent from their spontaneous mutants (Gao et  al., 2019). CRISPR/ Cas9 technology has become the main method to re-evaluate the Application of Gene Editing in Fruit Ripening important gene in fruit ripening through the generation of null mu- tant. The tomato nor mutant produces a new protein, 186-amino- The fleshy fruit undergoes a developmental process and ends with an acid protein (NOR186), which has a prohibitive function that affects irreversible maturation process. A lot of physiological, biochemical, ripening. The tomato nor mutant has previously been proven to be a and structural changes have taken place during the ripening process gain-of-function mutant, but the specific mechanism of action is still of fruit, as a result, it can attract more seed spreaders (Gapper et al., unclear (Wang et  al., 2019a). In nor natural mutants, the NOR186 2013). After the fruit reaches the optimum edible stage, the fruit will protein can still enter the nucleus, which can bind but cannot acti- slowly deteriorate and the fruit quality will be reduced. Therefore, vate the promoters of the key genes SlACS2, SlGgpps2, and SlPL for the regulation of fruit ripening has become the focus of many scien- ethylene biosynthesis, carotenoid accumulation, and fruit softening. tists (Martín-Pizarro and Posé, 2018). The activation effect of NOR protein on the above-mentioned pro- RIPENING INHIBITOR gene (RIN) belongs to the MADS-box moter will be inhibited by NOR186. The above research results fur- gene family and mutations of this gene can inhibit the ripening of ther prove that the nor natural mutant is a gain-of-function mutant, tomato fruits (Vrebalov et al., 2002). RIN is thought to be the main and the truncated protein NOR186 produced by the mutation of the regulatory gene in the maturation of tomato fruits and activates NOR gene has a dominant negative function (Gao et al., 2020). A re- and promotes all physiological processes associated with ripening, cent study indicated that VIGS technology can mediate nor-like1 silen- including colour, hardness, and flavour (Fujisawa et  al., 2013). The cing, which can suppress tomato fruit ripening. The inactive mutant of RIN gene is knocked out in tomato by CRISPR/Cas9 system and the nor-like1 was obtained through CRISPR/Cas9 technology, which de- rin mutant is analysed (Ito et al., 2015). The rin knockout mutation layed fruit ripening and inhibited ethylene, carotenoid synthesis, and does not affect the initiation of ripening and exhibits moderate red fruit softening. The above results indicate that nor-like1 gene plays a colouring, which demonstrates that RIN gene is not required for the positive regulatory role in tomato fruit ripening (Gao et al., 2018). ripening initiation in fruit (Ito et al., 2017). The rin mutant can lead According to reports, epigenetic modification and fruit ripening to ripening failure, which is caused by the deletion part of the DNA are inseparable. It has been found that the early epigenetic mark fragment between rin and the adjacent gene MACROCALYX (MC) is DNA cytosine methylation in the plant genome, which respond (Vrebalov et al., 2002). Due to the partial deletion of DNA fragments, to external environmental stress, regulate gene expression, and sta- the transcription factor MADS-RIN and MADS-MC are fused to bilize the genome (Chen et  al., 2018b). SlDML2 is closely related form the function of the fusion protein RIN–MC. The RIN–MC fu- to Arabidopsis DNA demethylase gene ROS1. SlDML2 knockout sion protein encodes a new transcription factor, which regulates the mutants were obtained by the CRISPR/Cas9 system, which inhibits expression of downstream genes and inhibits fruit ripening, which fruit ripening (Zhou et al., 2019). has a negative regulatory effect. (Vrebalov et  al., 2002; Ito et  al., In summary, compared with traditional knockdown technology, 2017; Li et  al., 2018d). Osorio et  al. (2020) reported that tomato mutants obtained by CRISPR/Cas technology can lead to unex- breeders use RIN mutation to acquire improved hybrids which exert pected weak phenotypes, which indicate that the compensation a negative impact on tomato flavour. Li et al. (2020) pointed out that mechanism in the body may obscure protein function. This requires RIN-deficient fruits obtained by CRISPR/Cas9 technology can reduce us to re-evaluate the model of the regulation of ripening, which may ethylene production and affect the synthesis of volatile substances involve a complex network of redundant components (Wang et al., and carotenoids. The low ethylene production is due to the fact that 2020). This reminds us to carefully design experimental programs RIN-deficient fruits cannot induce the production of ethylene in the when using CRISPR/Cas technology to evaluate other mechanisms. autocatalytic system-2. They also lack volatiles and carotenoids and transcripts related to these pathways. Meanwhile, Li et  al. (2020) supported that the fruit ripening process requires the participation of Gene Editing in Fruit Bioactive Compounds ERFs, RIN, and ethylene. Ethylene initiates the maturation of green fruit and affects the expression of RIN and other factors, which com- Many natural biologically active substances in fresh fruits have plete the whole ripening process of fruits (Li et al., 2020). anti-inflammatory, anti-cancer, anti-oxidation, and other physiological APETALA2a (AP2a), NON-RIPENING (NOR), and activities. Lycopene, carotenoid, anthocyanin, and gamma-aminobu- FRUITFULL (FUL1/TDR4 and FUL2/MBP7) play important roles tyric acid (GABA) are the main functional factors in fresh fruits. in fruit ripening (Mordy et  al., 1998; Chung et  al., 2010; Bemer Therefore, enhanced accumulation of bioactive substances has been et  al., 2012). But these results were derived from analysis of the focussed on by numerous studies (Amish et al., 2015). Downloaded from https://academic.oup.com/fqs/article/4/4/159/5940658 by DeepDyve user on 22 December 2020 162 X. Xu et al., 2020, Vol. 4, No. 4 Carotenoids play a protective role in ROS-mediated disorders Pectate lyases (PL) is an important component of pectinase. It is a and photosensitive or eye-related disorders. Carotenoids are mostly depolymerase that can degrade plant cell walls and lead to the soft- C40 terpenoids, which affect the growth, development, and matur- ening and even death of plant tissues (Uluisik and Seymour, 2020). ation of plants and improve the oxidative stability of poultry prod- The mutation of tomato PL gene is induced by CRISPR/cas9, which ucts such as egg and meat (Domonkos et  al., 2013; Nisar et  al., increases the firmness of the fruit and prolongs the shelf life of the 2015; Nabi et al., 2020). fruit, without negatively affecting other aspects of fruit ripening Lycopene is an acyclic carotenoid and red pigment that is abun- (Uluisik et al., 2016). dant in many ripening fruits. Lycopene reduces the risk of a variety Many ripening spontaneous mutants such as rin, nor, crn, and alc of tumours, including prostate cancer, and cardiovascular disease (Li can prolong storage time. The alc mutant has one base pair mutation and Xu, 2014; Tang et  al., 2014). In the process of fruit ripening, of NOR gene, resulting in nonsynonymous amino acid change (Yu lycopene is decreased due to the conversion to β-carotene and et al., 2017). Compared with the rin and nor mutants, the alc muta- α-carotene. In tomato, Li et al. (2018c) knocked out SGR1, LCY-E, tion not only prolongs shelf life, but also has better flavour and better BLC, LCY-B1, and LCY-B2 by the CRISPR/Cas9 method, which disease resistance. Tomato ALC gene mutation is obtained by using inhibited the conversion of lycopene and increased the lycopene the CRISPR/Cas9 method through HDR recombination pathway. content in the fruit about 5.1 times. During tomato fruit ripening, The alc homozygous mutant without T-DNA insertion exhibits im- phytoene synthase 1 (PSY1) is involved in the formation of lycopene proved storage time and prolonged shelf life (Yu et al., 2017). (Fray and Grierson,1993; Giorio et al., 2008). Gene editing of PSY1 was performed using CRISPR/Cas9 and the impaired PSY1 gene re- Application of Gene Editing in Fruit sults in the void of lycopene and yellow fruit, like yellow flesh mu- Colouration tant (D’Ambrosio et al., 2018). Lycopene desaturase (PDS) is an essential enzyme for the accumu- The difference in fruit colour is caused by the change of pigment. lation of lycopene and carotenoids (Bai et  al., 2016). The successful Genes affecting pigment synthesis can not only affect the bioactive mutations PDS1 and PDS2 of banana cv. Rasthali are generated by compounds, but also affect the colour of the fruit. In fruit and vege- the CRISPR/Cas9. Gene editing of the two genes resulted in the pre- table crops, colour is affecting consumer choice. For example, con- mature termination of PDS1 and PDS2 protein synthesis by inserting a sumers in Europe and the United States prefer red tomatoes, while termination codon into the gene sequences. The mutants exhibited de- Asians prefer pink tomatoes (Lin et al., 2014). The study of SlMYB12 creased chlorophyll and total carotenoid contents (Kaur et al., 2018). has proven to affect the accumulation of flavonoids. The mutation GABA, as a neuro-suppressant, has the functions of anti-fatigue, of SlMYB12 can produce pink tomato fruits (Ballester et al., 2010). sedation, and blood pressure regulation (Bachtiar et  al., 2015; The ant1 mutation is obtained by the CRISPR/cas9 system that can Takayama and Ezura, 2015). Research has proved that glutamate enhance the accumulation of anthocyanins and produce purple to- decarboxylase (GAD) catalysed the decarboxylation of glutamate to mato fruits (Čermák et al., 2015). PL, polygalacturonase 2a (PG2a), produce GABA (Akihiro et  al., 2008). GAD has a C-terminal self- and β-galactanase (TBG4) are tomato pectin degrading enzymes inhibiting region, and deletion of the domain promotes GAD activity that affect fruit ripening. Wang et  al. (2019a) obtained silent mu- (Takayama et al., 2015). In order to increase the content of GABA, tants of pl, pg2a, and tbg4. Interestingly, pg2a and tbg4 CRISPR Nonaka et  al. (2017) used the CRISPR/Cas9 method to delete the strains did not soften but affected the colour of the fruits (Wang C-terminal self-inhibitory domains of SlGAD2 and SlGAD3. The et al., 2019a). accumulation of GABA in mutant fruits increased by 7–15 times, As a widely used gene-editing technology, CRISPR/cas9 has great which affects the fruit size and yield in tomato (Nonaka et al., 2017). potential in the research of fruit colouration. Delila (Del) encodes a Previous studies have demonstrated that GABA transaminase transcription factor that has a basic helix-loop-helix (bHLH) domain (GABA-TP1, TP2, TP3), succinate semialdehyde dehydrogenase and Rosea1 (Ros1) encodes an MYB-related transcription factor (SSADH), and CAT9 are involved in GABA metabolism (Bao et al., (Goodrich et al., 1992; Schwinn et al., 2006). Butelli et al. (2008) ex- 2015; Snowden et  al., 2015). Li et  al. (2018a) successfully edited pressed the Del and Ros1 genes from snapdragon in tomato, which the five genes (GABA-TP1, GABA-TP2, GABA-TP3, SSADH, and the anthocyanin content of tomato fruit was significantly higher CAT9) in tomato genome by using pYLCRISPR/Cas9 vector, which than the accumulation of tomato anthocyanin previously reported. is a multi-locus gene knockout CRISPR/Cas9 system (Ma et  al., Engineering Del and Ros1 genes using CRISPR/Cas9 allows the cre- 2015). The multisite gene mutagenesis resulted in manipulated ation of novel mutants and holds great potential for studying the GABA metabolic pathways and significantly enhanced the GABA effect of transcription factors on fruit colouration. content (Li et al., 2018a). These studies show that CRISPR/Cas9 gene editing can be used Summary as an effective technique to modify bioactive compounds in fruits. The technology of gene editing is a powerful tool for functional gen- omics in improving the quality and commercial value of fruits. The Application of Gene Editing in Fruit Texture progresses on CRISPR/Cas9-mediated mutation involved in fruit Fruit texture is an indispensable factor in the study of fruit quality and ripening, fruit bioactive compounds, and fruit texture are summar- affects the commercial production of the fruit (Preeti et al., 2010). The ized in Table  1. The emergence of CRISPR/Cas9 technology pro- change of texture may lead to substantial decay of fruit in transporta- vides a new opportunity to accelerate plant molecular breeding. The tion and storage, which results in the development of typical diseases CRISPR/Cas9 technology used on fruit crops not only provided a during post-harvest storage and shelf life (Vicente et al., 2007). These shortcut to obtain high yield and good quality of fruit food, but texture changes are related to change the activity of many enzymes, also laid a solid foundation for fruit functional genomics research. which affect the structure of cell walls (Tucker et al., 2017). Therefore, An unavoidable problem with all gene-editing tools is the off-target fruit texture is closely related to fruit quality and shelf life. effect of non-specific site dissection of the genome. Off-target effects Downloaded from https://academic.oup.com/fqs/article/4/4/159/5940658 by DeepDyve user on 22 December 2020 Gene Editing Technology in Fruit Quality Improvement, 2020, Vol. 4, No. 4 163 Table 1. List of research on fruit improvement by using CRISPR/Cas gene-editing technology. Species Gene Gene function or phenotype Reference Tomato ALMT9 Decrease in malate content Ye et al. (2017) MPK20 Decrease in sugar content Chen et al. (2018a) ARF7 Parthenocarpic fruit Hu et al. (2018) GGP1 Ascorbic acid Li et al. (2018a) PG2a, TBG4 Fruit colour Wang et al. (2019b) SlMYB12 Fruit colour Ballester et al. (2010) ANT1 Fruit colour Čermák et al. (2015) CYCB Lycopene synthesis Zsögön et al. (2018) TBG4 Fruit firmness Wang et al. (2019b) CNR, NOR Fruit ripening Gao et al. (2019) RIN Fruit ripening Ito et al. (2017) SlEIN2, SlERFE1, SlARF2B, SlACS4 SlGRAS8, SlACS2 Fruit ripe and development Hu et al. (2019) lncRNA1459 Fruit ripening, lycopene, Li et al. (2018b) carotenoid biosynthesis SGR1, Blc, LCY-E, LCY-B1, LCY-B2 Increased lycopene content Li et al. (2018c) SlORRM4 Fruit ripening Yang et al. (2017) L1L4 Fruit metabolism Gago et al. (2017) SlAGL6 Parthenocarpic fruit Klap et al. (2017) RIN Fruit ripening Ito et al. (2015) AP2a, FUL1, FUL2, NOR Fruit ripening Wang et al. (2019b) SlDML2 Fruit ripening Zhou et al. (2019) SGR1, LCY-E, Blc, LCY-B1, LCY-B2 Lycopene synthesis Li et al. (2018c) PSY1 Lycopene synthesis D’Ambrosio et al. (2018) SlGAD2, SlGAD3 GABA content Nonaka et al. (2017) GABA-TP1, GABA content Ma et al. (2015) GABA-TP2, GABA-TP3, SSADH, CAT9 PL Fruit firmness Uluisik et al. (2016) ALC Long shelf life Yu et al. (2017) CLV3 Fruit size Zsögön et al. (2018) ENO Fruit size Yuste-Lisbona et al. (2020) Watermelon PDS Carotenoid biosynthesis Wang et al. (2019d) Banana PDS1, PDS2 Chlorophyll, Carotenoid Kaur et al. (2018) Apple IdnDH Biosynthesis of tartaric acid Osakabe et al. (2018) Grape VvPDS Albino phenotype Nakajima et al. (2017) Groundcherry ClV1 Fruit size Lemmon et al. (2018) Kiwifruit CEN Fruit development Varkonyi-Gasic et al. (2019) RIN, RIPENING INHIBITOR gene; PDS, Lycopene desaturase; PG2a, polygalacturonase 2a; TBG4, β-galactanase; CNR, colourless non- ripening; AP2a, APETALA2a; NOR, NON-RIPENING; PSY1, phytoene synthase 1; PL, Pectate lyases; CLV3, CLAVATA3; PDS, Lycopene desaturase. disrupt the expression of functional genes, affect gene functions, Conflict of Interest and eventually produce unpredictable adverse reactions (Guilinger The authors declare no conflict of interest. et al., 2014). Schaefer et al. (2017) published a peer-reviewed paper in which the authors reported that CRISPR-Cas9 caused unexpected References off-target changes in mice (Schaefer et  al., 2017). Therefore, gene- editing technology still needs to be further optimized to avoid off- Akihiro,  T., Koike,  S., Tani,  R., et  al. (2008). Biochemical mechanism on target and increase on-target editing efficiency. It is believed that GABA accumulation during fruit development in tomato. 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