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CRISPR基因编辑技术加速蔬菜作物遗传改良

花匠小妙招
2024-11-23 09:59

0 引言

利用遗传变异进行作物改良对植物育种至关重要。在植物育种历史中主要存在四种育种技术，分别为杂交育种，突变育种，转基因育种和基因编辑育种。由于杂交育种只能用来引入亲本基因组中已经存在的性状，遗传变异率低，突变育种中目的性状育种材料的鉴定需要投入巨大的工作量和时间，而转基因育种仍受限于市场监管的要求和一些民众对转基因商品的谨慎反对意见，因此利用基因编辑技术在不携带任何外源转基因的情况下直接对作物的基因进行编辑，实现精确地删除、替换或向基因组的目标位置中插入特定的序列以快速产生新的性状，为新品种培育和作物改良开辟了一条新的、更加快速的、更能被大众所接受的道路[1-2]。

由于存在序列特异性的DNA结合域或RNA序列，基因组编辑技术需要利用可以对特异DNA序列进行切割的核酸内切酶(EEN)[3]。核酸内切酶的发展历经三代，早期以锌指核酸酶(ZFN)和转录激活因子样效应物核酸酶(TALEN)为主，第三代则以规律性间隔的短回文序列重复簇(CRISPR)为核心[4]。许多研究表明，CRISPR的编辑效率高，且具有便于操作、成本低的优势，因此已经成为最广泛应用的基因编辑系统[5]。

CRISPR/Cas基因编辑系统自2013年出现以后，被广泛应用于农业、医学等多个领域，短短10年间前沿研究不断推进，学科交互日益深化，研究成果不断涌现。然而蔬菜作物种类繁多，性状丰富，关于CRISPR/Cas基因编辑系统在蔬菜作物遗传改良应用领域的研究进展和未来前沿热点探索的综述还不够系统、全面。本研究从基因编辑技术的组成、遗传转化效率的影响因素、提高遗传转化效率的方法以及目前基因编辑系统在主要蔬菜作物中的应用等方面进行综述，旨在系统、直观地展示CRISPR/Cas基因编辑体系应用的研究概况，以期为其在蔬菜作物中的深入研究及育种利用提供参考。

1 CRISPR基因编辑系统

1.1 CRISPR基因编辑系统的组成

构成CRISPR/Cas基因编辑系统的两部分分别是Cas核酸内切酶和向导RNA，传统的Cas9核酸内切酶特异性识别DNA上的NGG序列作为PAM序列，近些年发展出越来越多的Cas9变体并逐渐突破了NGG作为PAM序列的限制[6-7]。除Cas9核酸内切酶外，多种类型的核酸内切酶也相继被发现和应用，如V型中的Cpf1、C2c1、C2c2、C2c3、CasY和CasX，以及Ⅵ型中的C2c2、Cas13b、Cas13c等RNA引导的RNA内切酶，均在作物发展历程中起到了重要的作用[8-9]。

sgRNA由crRNA(CRISPR-derived RNA)和TracrRNA(trans-activating RNA)通过一段接头连接在一起形成，是CRISPR基因编辑体系中重要的一环，sgRNA的效率和特异性决定着CRISPR基因编辑的成败。CRISPR/Cas基因编辑可以耐受sgRNA和靶标序列之间一定程度的错配，导致脱靶编辑。BRAVO等[10]研究发现Cas9在12~14 mm错配位点（PAM远端第12~14位点）、18~20 mm错配位点（PAM远端第18~20位点）的活性是脱靶效应产生的关键因素。目前通常选择基因组特异性高、3'末端含有PAM序列、长度约20 bp、避免4个以上的T结尾、GC%含量为40%~60%的序列作为sgRNA序列。

1.2 CRISPR基因编辑载体的遗传转化

1.2.1 遗传转化效率的影响因素

农杆菌介导的遗传转化具有操作简单、重复性高、拷贝数少、实验成本低、可以将大量外源基因片段转移到宿主植物基因组中等优点，是目前植物中应用最广泛的遗传转化途径。农杆菌介导的植物转化的效率取决于植物-农杆菌之间的相互作用，此外植物基因型、外植体活力、农杆菌菌株类型、载体类型、选择系统和培养条件等都是农杆菌介导的植物转化成功的重要因素[11-12]。基因型依赖性是制约许多作物转基因研究及基因编辑研究的主要障碍，目前很多蔬菜作物的遗传转化体系仍依赖于特定的基因型，还有许多品种不适合进行转基因和基因编辑。比如在甘蓝中通常使用YL品种进行遗传转化，其他品种的转化效率远低于YL品种[13]。

1.2.2 基因共表达提高遗传转化效率

研究表明共表达生长调控基因可以显著提高转化效率。DEBERNARDI等[14]研究发现GRF4-GIF1嵌合体可大幅提高小麦、水稻以及柑橘等单子叶和双子叶植物的再生效率。WANG等[15]发现了一个与小麦植株再生相关的基因TaWOX5，TaWOX5基因可以大幅度提高小麦等植物的转化效率，克服基因型的限制。在蔬菜作物中，PAN等[16]测试了其它作物中可以提高组培遗传转化效率的生长调节基因，如BBM、WUS、GRF4-GIF1、GRF5、ipt等，发现多个生长调节基因都可以提高西瓜遗传转化效率，其中共表达GRF5基因可以将西农八号母本WWl50的遗传转化效率提高约40倍，达到24.73%。FENG等[17]通过过表达一个与内源性ClGRF4和ClGIF1融合的嵌合基因，突变ClGRF4基因内的miR396microRNA靶向位点进一步提高了西瓜的转化效率，建立了一种不依赖基因型的西瓜转化方法，在西瓜中实现了高效的基因编辑。LIAN等[18]发现PLT5、WIND1和WUS的表达可以提高金鱼草的遗传转化效率，PLT5和WUS的表达提高番茄遗传转化效率，PLT5基因可以在体外促进油菜和甜椒的出芽和转化效率，其中发育调控基因PLT5是提高植物转化效率的重要因子。

1.2.3 无需组织培养的遗传转化方法

寻找不需要组织培养或者能使遗传转化载体通过细胞壁的转化方法是植物转基因及基因编辑的主要研究方向[19]。MAHER等[20]报道在植物中同时表达DRs和基因编辑元件，在体细胞组织中重新诱导植物分生组织，不经过组培便获得了可以稳定遗传的转基因和基因编辑烟草。ELLISON等[21]将FT RNA与sgRNA融合后克隆到TRV载体中，FT促进sgRNA进入茎尖分生组织并以较高的效率产生可遗传的基因编辑，成功在烟草中起作用，不需要组织培养就可以获得稳定的遗传材料。LI等[22]开发了基于大麦条纹花叶病(BSMV)的sgRNA递送载体系统BSMV-sg，在小麦中实现了可遗传且无需组织培养和再生的基因组编辑。CAO等[23]研究开发了一个“切-浸-萌芽”(cut-dip-budding, CDB)递送系统，利用发根农杆菌侵染切后的根茎交界处，由茎产生转化根，再产生转化的植株，无需组织培养成功地实现了包括草本植物橡胶草和小冠花、块茎植物甘薯和木本植物臭椿等在内的多个物种的遗传转化。

2 CRISPR/Cas基因编辑系统在蔬菜中的应用

目前CRISPR基因编辑技术在作物改良中的应用主要集中在通过敲除一个或多个产生不利农艺性状的基因从而达到提高作物产量、品质和抗逆性的效果，对于快速创制蔬菜新品种，促进蔬菜产业可持续发展具有重大意义，相关研究进展主要集中在番茄、西瓜、甘蓝类蔬菜、胡萝卜、黄瓜等蔬菜作物中。

2.1 番茄

番茄是公认的进行功能基因组学和抗病研究的模式作物，因其具有重要的经济价值、中等大小的基因组、高质量的参考基因组、高效的遗传转化体系，是目前使用CRISPR/Cas系统进行功能研究最多的蔬菜作物[24]。果实的发育以及产量的提高是作物育种中最重要的一环。YUSTE-LISBONA等[25]的研究结果表明，通过对番茄中调控花分生组织活性的AP2/ERF转录因子SlENO进行编辑可以增加番茄果室的数目和果实大小，提高作物产量。ZSÖGÖN等[26]将农艺性状与野生番茄中的有用性状结合起来，通过编辑6个对番茄产量和生产力至关重要的基因：SELF-PRUNING、OVATE、FASCIATED、FRUIT WEIGHT 2.2、MULTIFLORA和LYCOPENE BETA CYCLASE得到了比野生番茄果实大小增加3倍，果实数量增加10倍的编辑植株。此外还有影响日中性和产量的LIN基因[27]、影响果实单性结实的SlAGL6基因和SlIAA9基因[28-29]、影响果实硬度的FIS1基因[30]等都通过基因编辑的手段验证了它们在果实发育过程中的功能。

在提高番茄果实风味方面，黄三文团队构建了番茄基因组变异图谱，获得了1200万个基因组变异的数据，鉴定了影响番茄33种风味物质的200多个主效遗传位点，并通过基因编辑获得了耐储运、风味佳的粉果番茄[31-32]。KAWAGUCHI等[33]通过敲除SlINVINH1基因，在保证果实重量不降低和植株生长不被破坏的情况下，成功地培育出了含糖量高的番茄株系。WANG等[34]利用CRISPR/Cas9技术获得了抑制番茄果实可溶性糖积累的两个基因(SlINVINH1和SlVPE5)的敲除系，并通过杂交获得了番茄果实甜度和加工品质提高的聚合品系。番茄和番茄产品是番茄红素的丰富来源，番茄红素的提高具有重要的育种价值[35]。NONAKA等[36]利用CRISPR/Cas9编辑SlGAD2和SlGAD3基因，使GABA(γ-氨基丁酸)积累增加7-15倍，同时对果实的大小和产量产生不同的影响。LI等[37]利用多重pYLCRISPR/Cas9系统对GABA代谢途径中5个基因（GABA-tp1、GABA-tp2、GABA-tp3、CAT9和SSADH）进行编辑，得到叶片和果实中GABA积累均显著增加的基因编辑株，四重突变株叶片中GABA含量比野生型高19倍，有效提高了番茄的营养价值。LI等[38]通过敲除类胡萝卜素代谢途径相关的5个基因（SGR1、LCY-E、Blc、LCY-B1和LCY-B2）成功创制了番茄红素含量增加约5.1倍的基因编辑番茄。除番茄红素外，影响类胡萝卜素合成的PDS基因[39]和PPSR1基因[40]以及影响花青素积累的SlAN2基因[41]也进行了编辑敲除和遗传转化，推进了番茄基因功能研究的进展。

由于驯化育种使的番茄栽培种质缺乏遗传多样性，阻碍了高抗性和耐受性新品种的开发。通过CRISPR/Cas9基因编辑技术可以使作物产生对特定病菌的抗性。NEKRASOV等[42]报道使用CRISPR/Cas9技术编辑SlMlo1基因在不到10个月的时间里培育出一种抗白粉病的非转基因番茄品种Tomelo。对番茄的SlPelo和SlMlo1基因进行编辑，介导番茄突变株产生了对黄曲叶病毒和白粉病的抗性[43]。LI等[44]通过多重CRISPR/Cas9系统编辑与形态、花和果实生产、抗坏血酸合成等相关的基因SP、SP5G、SlCLV3、SlWUS、和SlGGP1，将优异性状引入4个耐逆境野生番茄品种中，编辑过的植株保留了亲本的抗病性和耐盐性，其中两种具有细菌性斑点病抗性。TASHKANDI等[45]利用CRISPR/Cas9系统靶向编码病毒涂层蛋白(Coat protein，CP)或复制酶(Replicase，Rep)的序列，有效地干扰了病毒DNA的积累，提高了番茄对黄曲叶病毒TYLCV的抗性。ORTIGOSA等[46]利用CRISPR/Cas9系统编辑番茄中COR气孔共受体AtJAZ2的同源基因(SlJAZ2)，获得的编辑株完全阻止了由COR引起的气孔重新开放，减少了细菌通过气孔进入细胞的机会，增强了番茄对Pto DC3000菌株的抗性。VEILLET等[47]采用农杆菌介导的转化方法，对番茄乙酰乳酸合酶(ALS)基因中的目标胞苷碱基高效编辑，在番茄中获得了碱基编辑效率高达71%的抗除草剂植物。利用CRISPR/Cas9基因编辑系统，也证实了CCD8基因的突变可以用于提高番茄对根寄生杂草的抗性[48]。编辑番茄eIF4E1位点成功地增强了对PepMoV的抗性[49-50]。使用CRISPR/Cas9系统生成SlNPR1突变体，证实了SlNPR1可能参与番茄植株对干旱胁迫的响应[51]。

2.2 西瓜

西瓜是世界上最重要的瓜类作物之一[52]。由于编码类胡萝卜素生物合成途径关键酶的八氢番茄红素去饱和酶基因(PDS)被破坏引起的白化表型很容易被识别，因此在CRISPR/Cas基因编辑中常被选为靶向基因[1,53]。TIAN等[54]利用CRISPR/Cas9系统精准编辑西瓜原生质体细胞中的CIPDS基因产生了具有明显白化表型的突变体。ClATM1基因是第一个从西瓜中克隆的雄性不育基因，ZHANG等[55]通过CRISPR/Cas9介导的诱变验证了ClATM1基因调控花药发育的遗传功能。西瓜种子大小是西瓜育种中的一个重要农艺性状[56]。通过CRISPR/Cas9系统对西瓜ClBG1基因进行定向诱变，获得的clbg1突变体西瓜籽粒大小和重量均显著降低[56]。尖孢镰刀菌(FON)是西瓜枯萎病的主要致病因子，是全球西瓜育种的共同限制因素[57]。ZHANG等[58]利用CRISPR/Cas9系统敲除西瓜Clpsk1基因增强了西瓜对尖孢镰刀菌(FON)的抗性，证实了CRISPR/Cas9介导的基因编辑对西瓜育种改良的有效作用。TIAN等[59]通过CRISPR介导的胞嘧啶碱基编辑技术编辑ALS基因创制了抗除草剂西瓜。西瓜甜味以及鲜艳瓤色的出现是西瓜驯化过程中选择的重要特征，利用CRISPR/Cas9基因编辑系统对ClAGA2、ClSWEET3和ClTST2基因进行编辑，证实了西瓜甜蜜基因在促进西瓜果肉糖分积累中的关键作用，揭示了西瓜“甜蜜基因”驯化的秘密[60-61]。CRISPR/Cas基因编辑系统也为选育优质雌性西瓜系提供了理论和技术依据，ZHANG等[62]利用CRISPR/Cas9基因编辑系统对在心皮原基中特异表达、并与花发育早期心皮原基的败育有关的ClWIP1基因进行编辑获得了西瓜雌性系。褪黑素参与调节植物生长和对各种非生物胁迫的反应，利用CRISPR/Cas9系统敲除ClCOMT1(Cla97C07G144540)基因降低了西瓜愈伤组织中的褪黑素含量，证实了ClCOMT1基因在西瓜褪黑素生物合成中的重要作用[63]。

2.3 甘蓝类蔬菜

利用基因编辑系统，LAWRENSON等[64]以BolC.GA4为靶点，首次在甘蓝中进行基因编辑获得了预期的矮化表型。SUN等[1]首次报道了CRISPR/Cas9介导的芥蓝基因组编辑技术，通过编辑同源基因BaPDS1和BaPDS2，经农杆菌介导的遗传转化获得了包括双突变和单突变在内的多个不同突变类型的芥蓝转基因株系，且所有突变体都能观察到明显的白化表型。MA等[65]利用CRISPR/Cas9基因编辑系统结合内源性tRNA处理系统编辑八氢番茄红素脱氢基因BoPDS、自交不亲和决定基因BoSRK3以及雄性不育相关基因BoMS1成功获得了相应表型的甘蓝编辑株系。蜡质沉积与生物和非生物胁迫耐受性密切有关，对甘蓝生长发育具有十分重要的意义[66-67]。CAO等[68]构建基因编辑载体CRISPR-BoCER1，证实了BoCER1基因在甘蓝表皮蜡质生物合成中的重要作用。此外，以胡萝卜素异构酶基因(BoaCRTISO)为目标基因，利用CRISPR/Cas9系统进行基因编辑获得了类胡萝卜素和叶黄素含量均降低的黄色羽衣甘蓝突变体，促进了羽衣甘蓝的蔬菜品质改良[69]。

2.4 胡萝卜

KLIMEK-CHODACKA等[70]利用农杆菌介导的CRISPR/Cas9系统，在胡萝卜中实现了对黄酮-3-羟基化酶(F3H)基因的高效靶向突变，验证了该基因在胡萝卜花青素生物合成中的功能。以DcPDS和DcMYB113-like基因的4个位点作为靶点，敲除桔色胡萝卜“Kurodagosun”的DcPDS基因获得了白化胡萝卜，编辑紫胡萝卜“Deep purple”的DcMYB113-like基因得到了脱紫胡萝卜植株，成功将CRISPR/Cas9系统用于胡萝卜基因组的精确编辑和功能验证[71]。

2.5 黄瓜

CHANDRASEKARAN等[72]利用CRISPR/Cas9基因编辑技术，首次在黄瓜中创制出对黄瓜脉黄化病毒(CVYV)、小西葫芦黄花叶病毒(ZYMV)和番木瓜环斑花叶病毒(PRSV-W)具有抗性的eif4e非转基因突变体。HU等[73]进一步优化了CRISPR/Cas9系统，突变了CsWIP1(Csa4M290830)、CsVFB1(Csa4M641640)、CsMLO8(Csa5M623470)和CsGAD1(Csa5M348050)基因，证实了Cswip1基因在抑制黄瓜心皮发育中所起的作用，并从一个有商业价值的自交系中获得了非转基因黄瓜雌性系。果实的大小、形状以及棱瘤刺是影响黄瓜市场价值的重要品质性状。XIN等[74]通过创制SF1基因(Csa2G174140)以及ACS2基因(Csa1G580750)的黄瓜编辑株系，验证了SF1基因和ACS2基因分别在导致黄瓜短果表型产生以及黄瓜果实形成中的重要作用。YANG等[75]利用CRISPR/Cas9靶向TEN基因和ACO1基因，证实了这两个基因在卷须性状控制中的作用。ZHANG等[76]使用CRISPR/Cas9基因编辑系统敲除SF2基因使突变体枝条生长受到显著抑制，验证了该基因在控制分生细胞增殖方面的功能。WANG等[77]利用CRISPR/Cas9系统敲除HECATE2基因(CsHEC2)后，棱瘤刺密度降低，细胞分裂素(CTK)在果皮中的积累减少，说明CsHEC2基因促进棱瘤刺的形成。此外，ZHANG等[78]采用CRISPR/Cas9基因编辑技术和甲基磺酸乙酯(EMS)诱变技术确定了ACS1G基因在决定黄瓜雌花发育中的功能。

2.6 马铃薯

利用CRISPR/Cas9技术敲除马铃薯GBSS、SBE1或SBE2基因，获得了具有重要商业价值的支链淀粉马铃薯或短链减少、长链增加淀粉品质的马铃薯[79]。高水平甾体糖碱(SGA)的存在可能会导致苦味和潜在的不良影响，因此减少马铃薯块茎中的甾体糖碱(SGA)是培育优质马铃薯的关键。NAKAYASU等[80]将编码SGA生物合成过程甾体16α-羟化酶的St16DOX基因的多个gRNA与pMgP237-2A-GFP载体结合，敲除St16DOX基因，使马铃薯毛状根中积累的SGA全部消失，获得了无SGA毛状根的四倍体马铃薯。利用CRISPR/Cas9基因组编辑技术对S-RNase基因进行了定点突变，获得了自交亲和的二倍体马铃薯[81]。采用农杆菌介导的转化方法，高效编辑马铃薯中的乙酰乳酸合酶基因(StALS)，创制了除草剂抗性株系[47,82]。ZHAN等[83]利用CRISPR/Cas13a基因编辑系统创制的马铃薯转基因株系成功表现出了对马铃薯Y病毒(PVY)积累和病症表型的抑制作用。

2.7 其他蔬菜

白菜农艺性状改良和新品种创制在很长时间内都依赖于杂交、回交和分子标记辅助选择。导致这种现象的原因是白菜中缺乏高效成熟的遗传转化体系，大部分基因功能的验证都是利用拟南芥、烟草等模式植物进行的，这也在很大程度上限制了基础理论研究的深度。尽管存在困难，但目前利用CRISPR/Cas9系统在促进白菜品种改良方面还是取得了阶段性的成果。JUNG等[84]利用CRISPR/Cas9系统对BrSOC1的同源基因(BrSOC1s)进行定向诱变，并成功将晚开花性状导入精英品种“20”(2n=20)，证实了基因编辑技术可用于提高大白菜产量。JEONG等[85]设计了7个gRNA靶向白菜中FLC开花基因的同源基因，创制了不依赖于春化条件的早开花白菜，证实了多拷贝BraFLCs基因的功能。

在菊苣中，使用sgRNAs和CRISPR/Cas9基因编辑系统对菊苣进行了高效的多重基因组编辑，成功地敲除了菊苣毛状根再生植株和原生质体中的CiPDS基因，产生了白化株系[86]。

在莴苣中，LsNCED4基因也被用于编辑以验证CRISPR/Cas9基因编辑系统的适用性和基因功能，突变体种子萌发的最高温度大大提高[87]。

在甜瓜和南瓜中，典型的PDS基因也被编辑以验证CRISPR/Cas9介导的甜瓜基因组编辑可行性，农杆菌介导转化后CmPDS基因的两个靶点发生了突变，显现出白化表型[88]。XIN等[89]以受体激酶基因ERECTA家族的同源基因（甜瓜中的MELO3C016916基因及南瓜中的CmoCh09G003660基因和CmoCh01G017570基因）为靶点，创制了植株结构紧凑，节间更短的甜瓜和南瓜。通过CRISPR/Cas9基因编辑技术敲除南瓜RBOHD基因，证实了该基因在H2O2和K+含量积累中的积极作用[90]。CRISPR/Cas9基因编辑技术同样也被应用于编辑南瓜砧木上的钠依赖性高亲和力K+转运蛋白1基因(CmoHKT1;1)，将根转化方法与嫁接相结合，在砧木上进行基因编辑，证实了该基因在耐盐性方面的作用[91]。

在茄子中，采用基于CRISPR/Cas9的基因编辑方法，将Cas9蛋白定位到3个PPO基因（SmelPPO4、SmelPPO5和SmelPPO6）的保守区域，敲除了这3个目标PPO基因，编辑株系中的多酚氧化酶活性降低，保持了较高的多元酚含量，果肉在切割后的酶促褐变减少，为低酶促褐变型茄子育种开辟了新的道路[92]。

在辣椒中，使用CRISPR/Cas9-RNP和CRISPR/LbCpf1-RNP成功对辣椒品种CM334和Dempsey原生质体的白粉病抗性基因CaMLO2（拟南芥AtMLO2基因的同源基因）进行编辑，促进了基因编辑在辣椒品种改良中的发展进程[93-94]。此外，利用CRISPR/Cas9基因编辑系统编辑辣椒炭疽病易感基因CaERF28，增强了辣椒对炭疽病的抗性[95]。

在生菜中，对编码维生素C生物合成关键酶基因LsGGP2的ORF进行编辑，提高了生菜中的抗坏血酸含量和生菜对氧化胁迫的耐受性[96]。

3 展望

通过对CRISPR基因编辑系统的组成、遗传转化效率以及目前基因编辑系统在主要蔬菜作物遗传改良中的应用进行总结、分析，得出以下结论：

（1）CRISPR基因编辑系统目前仍存在一些不可避免的缺陷。一方面，育种家选择的许多农艺性状都是多基因的，尤其是很多品质相关的性状受多个QTLs共同控制，而CRISPR/Cas基因编辑系统大多更适合针对一个或几个遗传位点，编辑单个基因可能不会引起显著的表型变化[97]。另一方面，CRISPR/Cas基因编辑系统创制功能丧失突变体比创制功能获得突变体容易得多。作为一款强大的工具，基因编辑技术可以实现的还有很多，未来在碱基编辑器和引导编辑器等新型基因编辑工具的有效利用下高效、快速获得功能获得突变体也将成为研究的重点。比如V226F和K435N这两个控制西瓜红瓤基因ClLCYB的点突变。如果可以在西瓜中实现碱基的任意替换，那么就可以通过对LCYB基因进行CRISPR碱基编辑，通过番茄红素含量增加的表型验证基因功能[98-99]。此外，从已经确定的基因靶点入手，进一步拓展到控制表型的新基因、调控元件和染色质修饰，从而实现表观基因组编辑在蔬菜中的应用，阐明蔬菜基因表达调控机制也将成为未来CRISPR基因编辑系统的研究热点[100-101]。

（2）植物转化和再生系统仍然是植物基因组编辑的瓶颈，导致许多蔬菜作物遗传转化和基因编辑的可重复性和效率低。遗传转化系统是作物遗传改良、基因结构及功能分析最重要的平台之一，特定的作物品种基因型起到很重要的作用，需要进行广泛的筛选以选择易于遗传转化的基因型品种。开发高效的植物再生系统也是蔬菜基因编辑发展的重要方向。基因编辑系统传递方法也很重要，不仅影响转化效率，而且影响脱靶效应和后期调控目的。最初的CRISPR介导的基因组编辑方法依赖于传递编码Cas和sgRNAs的质粒或病毒载体，由于这些载体对一些大的Cas核酸酶的包装效果不佳，不易于在体内传送，因此需要开发新的CRISPR/Cas系统的传递方法。

（3）开发兼具低脱靶效应、高切割速率、新型、多功能的基因编辑系统十分必要。通过优化sgRNA或人工修改Cas9蛋白，研究出多种低脱靶效应的Cas9突变体，降低了CRISPR/Cas9基因编辑系统的脱靶效率，但突变体在靶位点的切割速率也有明显降低[102-103]，因此促进基因编辑工具的改进和迭代也将是未来的研究热点。此外，植物细胞器中的基因编辑也是未来蔬菜基因编辑技术发展的重要方向。过去由于细胞器基因组具有包裹细胞器、阻止大多数核酸进入的双膜结构，并且缺乏合适的工具来靶向这些细胞器中的DNA，使得植物细胞器基因组编辑进展缓慢[104-105]。目前在酵母线粒体和衣藻叶绿体中实现了CRISPR介导的细胞器基因组编辑[104]。KANG等[105]在叶绿体中实现了DNA自由碱基编辑，获得了对链霉素和大光霉素耐药的生菜愈伤组织和植株，这些进展为蔬菜细胞器基因组编辑的进一步发展带来希望，未来更多的蔬菜作物将实现细胞器基因组的精准编辑以及新基因的引入。

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崔慧琳, 李志远, 方智远, 等. 结球甘蓝自交系YL-1的高效遗传转化体系的建立及应用[J]. 园艺学报, 2019, 46(2):345-355.

为了筛选结球甘蓝高效遗传转化受体材料，以高代自交系YL-1、21-3、12J35、650为试材，比较不同材料具柄子叶和下胚轴的再生频率差异。在4份供试材料中，YL-1为最优转化受体，再生频率显著高于其他3份材料；YL-1具柄子叶和下胚轴的再生频率无显著差异，均可用于遗传转化。进一步对YL-1遗传转化过程中光照强度、Basta除草剂浓度、农杆菌侵染浓度等关键影响因素进行优化，发现光照强度为4 000 lx时更有利于不定芽诱导；除草剂最适筛选浓度为8 mg · L-1；农杆菌侵染浓度OD600 = 0.3侵染8 min，YL-1抗性芽再生频率最高。基于建立的高效遗传转化体系，共获得47株抗除草剂植株，PCR检测显示其中12株为阳性，初步表明bar基因已转化到甘蓝中，转化率达到2.18%。利用转录组测序技术对2株PCR阳性材料进行基因表达分析，均检测到bar基因高效表达。甘蓝高代自交系YL-1高效遗传转化体系的建立可为今后结球甘蓝基因功能鉴定及重要农艺性状的改良提供基础。

TRICOLI D M

ERCOLI M F

, et al. A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants[J]. Nat biotechnol, 2020, 38(11):1274-1279.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.

SHI L

LIANG X N

, et al. The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation[J]. Nat plants, 2022, 8(2):110-117.

Although great progress has been achieved regarding wheat genetic transformation technology in the past decade, genotype dependency, the most impactful factor in wheat genetic transformation, currently limits the capacity for wheat improvement by transgenic integration and genome-editing approaches. The application of regeneration-related genes during in vitro culture could potentially contribute to enhancement of plant transformation efficiency. In the present study, we found that overexpression of the wheat gene TaWOX5 from the WUSCHEL family dramatically increases transformation efficiency with less genotype dependency than other methods. The expression of TaWOX5 in wheat calli prohibited neither shoot differentiation nor root development. Moreover, successfully transformed transgenic wheat plants can clearly be recognized based on a visible botanic phenotype, relatively wider flag leaves. Application of TaWOX5 improved wheat immature embryo transformation and regeneration. The use of TaWOX5 in improvement of transformation efficiency also showed promising results in Triticum monococcum, triticale, rye, barley and maize.© 2022. The Author(s), under exclusive licence to Springer Nature Limited.

CHENG Z T

HAN Z G

, et al. Efficient genetic transformation and CRISPR/Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators[J]. J zhejiang univ sci b, 2022, 23(4):339-344.

XIAO L

HE Y Z

, et al. Highly efficient, genotype-independent transformation and gene editing in watermelon (Citrullus lanatus) using a chimeric ClGRF4-GIF1 gene[J]. J integr plant biol, 2021, 63(12):2038-2042.

NGUYEN C D

LIU L

, et al. Application of developmental regulators to improve in planta or in vitro transformation in plants[J]. Plant biotechnol j, 2022, 20(8):1622-1635.

KAEPPLER S M

OLHOF P

. Genetic instability of plant tissue cultures: breakdown of normal controls[J]. Proc natl acad sci U S A, 1994, 91(12):5222-5226.

NASTI R A

VOLLBRECHT M

, et al. Plant gene editing through de novo induction of meristems[J]. Nat biotechnol, 2020, 38(1):84-89.

Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing.

NAGALAKSHMI U

GAMO M E

, et al. Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs[J]. Nat plants, 2020, 6(6):620-624.

An in planta gene editing approach was developed wherein Cas9 transgenic plants are infected with an RNA virus that expresses single guide RNAs (sgRNAs). The sgRNAs are augmented with sequences that promote cell-to-cell mobility. Mutant progeny are recovered in the next generation at frequencies ranging from 65 to 100%; up to 30% of progeny derived from plants infected with a virus expressing three sgRNAs have mutations in all three targeted loci.

HU J C

SUN Y

, et al. Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture[J]. Mol plant, 2021, 14(11):1787-1798.

Genome editing provides novel strategies for improving plant traits, but relies on current genetic transformation and plant regeneration procedures, which can be inefficient. We have engineered a barley stripe mosaic virus (BSMV)-based sgRNA delivery vector (BSMV-sg) that is effective in performing heritable genome editing in Cas9-transgenic wheat plants. Mutated progenies were present in the next generation at frequencies ranging from 12.9% to 100% in three different wheat varieties, and 53.8% to 100% of mutants were virus-free. We also achieved multiplex mutagenesis in progeny using a pool of BSMV-sg vectors harboring different sgRNAs. Furthermore, we devised a virus-induced transgene-free editing procedure (VITF-Edit) to generate Cas9-free wheat mutants by crossing BSMV-infected Cas9-transgenic wheat pollen with wild-type wheat. Our study provides a robust, convenient and tissue culture-free approach for genome editing in wheat through virus infection.Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.

XIE H T

SONG M L

, et al. Cut-dip-budding delivery system enables genetic modifications in plants without tissue culture[J]. The innovation, 2022, 4(1):100345.

M MAHMOUD L

S MORAES T

, et al. Development of improved fruit, vegetable, and ornamental crops using the CRISPR/Cas9 genome editing technique[J]. Plants (Basel), 2019, 8(12):601.

FERNÁNDEZ-LOZANO A

PINEDA B

, et al. ENO regulates tomato fruit size through the floral meristem development network[J]. Proc natl acad sci U S A, 2020, 117(14):8187-8195.

ČERMÁK T

NAVES E R

, et al. De novo domestication of wild tomato using genome editing[J]. Nat biotechnol, 2018, 36:1211-1216.

Breeding of crops over millennia for yield and productivity ) has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost 2. We devised a CRISPR-Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. We report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium. Engineered S. pimpinellifolium morphology was altered, together with the size, number and nutritional value of the fruits. Compared with the wild parent, our engineered lines have a threefold increase in fruit size and a tenfold increase in fruit number. Notably, fruit lycopene accumulation is improved by 500% compared with the widely cultivated S. lycopersicum. Our results pave the way for molecular breeding programs to exploit the genetic diversity present in wild plants.

{{custom_citation.annotation}4}https://doi.org/{{custom_citation.annotation}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.annotation}0}{{custom_ref.label}8}本文引用 [{{custom_ref.label}3}]摘要{{custom_ref.label}2}[27]SOYK S

LEMMON Z H

OVED M

, et al. Bypassing negative epistasis on yield in tomato imposed by a domestication gene[J]. Cell, 2017, 169(6):1142-1155.

Selection for inflorescence architecture with improved flower production and yield is common to many domesticated crops. However, tomato inflorescences resemble wild ancestors, and breeders avoided excessive branching because of low fertility. We found branched variants carry mutations in two related transcription factors that were selected independently. One founder mutation enlarged the leaf-like organs on fruits and was selected as fruit size increased during domestication. The other mutation eliminated the flower abscission zone, providing "jointless" fruit stems that reduced fruit dropping and facilitated mechanical harvesting. Stacking both beneficial traits caused undesirable branching and sterility due to epistasis, which breeders overcame with suppressors. However, this suppression restricted the opportunity for productivity gains from weak branching. Exploiting natural and engineered alleles for multiple family members, we achieved a continuum of inflorescence complexity that allowed breeding of higher-yielding hybrids. Characterizing and neutralizing similar cases of negative epistasis could improve productivity in many agricultural organisms. VIDEO ABSTRACT.Copyright © 2017 Elsevier Inc. All rights reserved.

{{custom_ref.label}1}https://doi.org/{{custom_citation.content}9}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.content}7}{{custom_citation.content}5}本文引用 [{{custom_citation.content}0}]摘要{{custom_citation.doi}9}[28]KLAP C

YESHAYAHOU E

BOLGER A M

, et al. Tomato facultative parthenocarpy results from SlAGAMOUS-LIKE 6 loss of function[J]. Plant biotechnol j, 2017, 15(5):634-647.

The extreme sensitivity of the microsporogenesis process to moderately high or low temperatures is a major hindrance for tomato (Solanum lycopersicum) sexual reproduction and hence year-round cropping. Consequently, breeding for parthenocarpy, namely, fertilization-independent fruit set, is considered a valuable goal especially for maintaining sustainable agriculture in the face of global warming. A mutant capable of setting high-quality seedless (parthenocarpic) fruit was found following a screen of EMS-mutagenized tomato population for yielding under heat stress. Next-generation sequencing followed by marker-assisted mapping and CRISPR/Cas9 gene knockout confirmed that a mutation in SlAGAMOUS-LIKE 6 (SlAGL6) was responsible for the parthenocarpic phenotype. The mutant is capable of fruit production under heat stress conditions that severely hamper fertilization-dependent fruit set. Different from other tomato recessive monogenic mutants for parthenocarpy, Slagl6 mutations impose no homeotic changes, the seedless fruits are of normal weight and shape, pollen viability is unaffected, and sexual reproduction capacity is maintained, thus making Slagl6 an attractive gene for facultative parthenocarpy. The characteristics of the analysed mutant combined with the gene's mode of expression imply SlAGL6 as a key regulator of the transition between the state of 'ovary arrest' imposed towards anthesis and the fertilization-triggered fruit set.© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

ABE C

WATANABE T

, et al. Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9[J]. Sci rep, 2017, 7:507.

Parthenocarpy in horticultural crop plants is an important trait with agricultural value for various industrial purposes as well as direct eating quality. Here, we demonstrate a breeding strategy to generate parthenocarpic tomato plants using the CRISPR/Cas9 system. We optimized the CRISPR/Cas9 system to introduce somatic mutations effectively into SlIAA9-a key gene controlling parthenocarpy-with mutation rates of up to 100% in the T0 generation. Furthermore, analysis of off-target mutations using deep sequencing indicated that our customized gRNAs induced no additional mutations in the host genome. Regenerated mutants exhibited morphological changes in leaf shape and seedless fruit-a characteristic of parthenocarpic tomato. And the segregated next generation (T1) also showed a severe phenotype associated with the homozygous mutated genome. The system developed here could be applied to produce parthenocarpic tomato in a wide variety of cultivars, as well as other major horticultural crops, using this precise and rapid breeding technique.

{{custom_citation.doi}5}https://doi.org/{{custom_citation.doi}3}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.doi}1}{{custom_citation.pmid}9}本文引用 [{{custom_citation.pmid}4}]摘要{{custom_citation.pmid}3}[30]LI R

SUN S

WANG H J

, et al. FIS1 encodes a GA2-oxidase that regulates fruit firmness in tomato[J]. Nat commun, 2020, 11(1):5844.

Fruit firmness is a target trait in tomato breeding because it facilitates transportation and storage. However, it is also a complex trait and uncovering the molecular genetic mechanisms controlling fruit firmness has proven challenging. Here, we report the map-based cloning and functional characterization of qFIRM SKIN 1 (qFIS1), a major quantitative trait locus that partially determines the difference in compression resistance between cultivated and wild tomato accessions. FIS1 encodes a GA2-oxidase, and its mutation leads to increased bioactive gibberellin content, enhanced cutin and wax biosynthesis, and increased fruit firmness and shelf life. Importantly, FIS1 has no unfavorable effect on fruit weight or taste, making it an ideal target for breeders. Our study demonstrates that FIS1 mediates gibberellin catabolism and regulates fruit firmness, and it offers a potential strategy for tomato breeders to produce firmer fruit.

{{custom_citation.pmid}2}https://doi.org/{{custom_citation.pmid}0}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.pmid}8}{{custom_citation.pmid}6}本文引用 [{{custom_citation.pmid}1}]摘要{{custom_citation.pmid}0}[31]TIEMAN D

ZHU G T

RESENDE MFR J

, et al. A chemical genetic roadmap to improved tomato flavor[J]. Science, 2017, 355(6323):391-394.

Modern commercial tomato varieties are substantially less flavorful than heirloom varieties. To understand and ultimately correct this deficiency, we quantified flavor-associated chemicals in 398 modern, heirloom, and wild accessions. A subset of these accessions was evaluated in consumer panels, identifying the chemicals that made the most important contributions to flavor and consumer liking. We found that modern commercial varieties contain significantly lower amounts of many of these important flavor chemicals than older varieties. Whole-genome sequencing and a genome-wide association study permitted identification of genetic loci that affect most of the target flavor chemicals, including sugars, acids, and volatiles. Together, these results provide an understanding of the flavor deficiencies in modern commercial varieties and the information necessary for the recovery of good flavor through molecular breeding.Copyright © 2017, American Association for the Advancement of Science.

{{custom_citation.url}9}https://doi.org/{{custom_citation.url}7}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.url}5}{{custom_citation.url}3}本文引用 [{{custom_citation.url}8}]摘要{{custom_citation.url}7}[32]ZHU G T

WANG S C

HUANG Z J

, et al. Rewiring of the fruit metabolome in tomato breeding[J]. Cell, 2018, 172(1-2):249-261.

Humans heavily rely on dozens of domesticated plant species that have been further improved through intensive breeding. To evaluate how breeding changed the tomato fruit metabolome, we have generated and analyzed a dataset encompassing genomes, transcriptomes, and metabolomes from hundreds of tomato genotypes. The combined results illustrate how breeding globally altered fruit metabolite content. Selection for alleles of genes associated with larger fruits altered metabolite profiles as a consequence of linkage with nearby genes. Selection of five major loci reduced the accumulation of anti-nutritional steroidal glycoalkaloids in ripened fruits, rendering the fruit more edible. Breeding for pink tomatoes modified the content of over 100 metabolites. The introgression of resistance genes from wild relatives in cultivars also resulted in major and unexpected metabolic changes. The study reveals a multi-omics view of the metabolic breeding history of tomato, as well as provides insights into metabolome-assisted breeding and plant biology.Copyright © 2017 Elsevier Inc. All rights reserved.

{{custom_citation.url}6}https://doi.org/{{custom_citation.url}4}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.url}2}{{custom_citation.url}0}本文引用 [{{custom_ref.citedCount>0}5}]摘要{{custom_ref.citedCount>0}4}[33]KAWAGUCHI K

TAKEI-HOSHI R

YOSHIKAWA I

, et al. Functional disruption of cell wall invertase inhibitor by genome editing increases sugar content of tomato fruit without decrease fruit weight[J]. Sci rep, 2021, 11(1):21534.

Sugar content is one of the most important quality traits of tomato. Cell wall invertase promotes sucrose unloading in the fruit by maintaining a gradient of sucrose concentration between source leaves and fruits, while invertase inhibitor (INVINH) regulates this process. In this study, knock-out of cell wall INVINH in tomato (SlINVINH1) was performed by genome editing using, CRISPR/Cas9 and Target-AID technologies. Most of the genome-edited lines set higher soluble solid content (SSC) fruit than the original cultivar 'Suzukoma', while fruit weight was different among the genome-edited lines. From these genome-edited lines, three lines (193-3, 199-2, and 247-2), whose SSC was significantly higher than 'Suzukoma' and fruit weight were almost the same as the original cultivar, were selected. The fruit weight and overall plant growth of the two lines were comparable to those of the original cultivar. In contrast, the fructose and glucose contents in the mature fruits of the two lines were significantly higher than those of the original cultivar. The mature fruits of genome edited line 193-3 showed the highest sugar content, and the fructose and glucose contents were 29% and 36% higher than that of the original cultivar, respectively. Whole genome sequence data showed no off-target mutations in the genome-edited lines. Non-target metabolome analysis of mature fruits revealed that fructose was the highest loading factor in principal component analysis (PCA) between the genome-edited line and the original cultivar, and no unexpected metabolites appeared in the genome-edited line. In this study, we succeeded in producing tomato lines with high sugar content without a decrease in fruit weight and deterioration of plant growth by knock-out of SlINVINH1 using genome editing technology. This study showed that functional disruption of SlINVINH1 is an effective approach to produce tomato cultivars with high sugar content.© 2021. The Author(s).

{{custom_ref.citedCount>0}3}https://doi.org/{{custom_ref.citedCount>0}1}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citationIndex}9}{{custom_citationIndex}7}本文引用 [{{custom_citationIndex}2}]摘要{{custom_citationIndex}1}[34]WANG B K

LI N

HUANG S Y

, et al. Enhanced soluble sugar content in tomato fruit using CRISPR/Cas9-mediated SlINVINH1 and SlVPE5 gene editing[J]. Peerj, 2021, 9:e12478.

{{custom_citationIndex}0}https://doi.org/{{custom_ref.citationList}8}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.citationList}6}{{custom_ref.citationList}4}本文引用 [{{custom_ref.id}9}]摘要{{custom_ref.id}8}[35]WALALLAWITA U S

WOLBER F M

ZIV-GAL A

, et al. Potential role of lycopene in the prevention of postmenopausal bone loss: evidence from molecular to clinical studies[J]. Int j mol sci, 2020, 21(19):7119.

{{custom_ref.id}7}https://doi.org/{{custom_ref.id}5}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.id}3}{{custom_ref.id}1}本文引用 [{{custom_ref.citedCount}6}]摘要{{custom_ref.citedCount}5}[36]NONAKA S

ARAI C

TAKAYAMA M

, et al. Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis[J]. Sci rep, 2017, 9(1):19822.

{{custom_ref.citedCount}4}https://doi.org/{{custom_ref.citedCount}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.citedCount}0}{{custom_citation.annotation}8}本文引用 [{{custom_citation.annotation}3}]摘要{{custom_citation.annotation}2}[37]LI R

LI R

LI X

, et al. Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum[J]. Plant biotechnol j, 2018, 16(2):415-427.

{{custom_citation.annotation}1}https://doi.org/{{custom_fund}9}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_fund}7}{{custom_fund}5}本文引用 [{{custom_fund}0}]摘要{{custom_citation.annotation}}[38]LI X D

WANG Y N

CHEN S

, et al. Lycopene is enriched in tomato fruit by CRISPR/Cas9-mediated multiplex genome editing[J]. Front plant sci, 2018, 9:559.

Numerous studies have been focusing on breeding tomato plants with enhanced lycopene accumulation, considering its positive effects of fruits on the visual and functional properties. In this study, we used a bidirectional strategy: promoting the biosynthesis of lycopene, while inhibiting the conversion from lycopene to beta- and alpha-carotene. The accumulation of lycopene was promoted by knocking down some genes associated with the carotenoid metabolic pathway. Finally, five genes were selected to be edited in genome by CRISPR/Cas9 system using Agrobacterium tumefaciens-mediated transformation. Our findings indicated that CRISPR/Cas9 is a site-specific genome editing technology that allows highly efficient target mutagenesis in multiple genes of interest. Surprisingly, the lycopene content in tomato fruit subjected to genome editing was successfully increased to about 5.1-fold. The homozygous mutations were stably transmitted to subsequent generations. Taken together, our results suggest that CRISPR/Cas9 system can be used for significantly improving lycopene content in tomato fruit with advantages such as high efficiency, rare off-target mutations, and stable heredity.

BHATTACHARYA A

CHOUDHARY S

, et al. Demonstration of CRISPR-cas9-mediated pds gene editing in a tomato hybrid parental line[J]. Indian j genet, 2018, 78:132-137.

WANG Y

WANG W

, et al. Ubiquitination of phytoene synthase 1 precursor modulates carotenoid biosynthesis in tomato[J]. Commun biol, 2020, 3(1):730.

Carotenoids are natural pigments that are indispensable to plants and humans, whereas the regulation of carotenoid biosynthesis by post-translational modification remains elusive. Here, we show that a tomato E3 ubiquitin ligase, Plastid Protein Sensing RING E3 ligase 1 (PPSR1), is responsible for the regulation of carotenoid biosynthesis. PPSR1 exhibits self-ubiquitination activity and loss of PPSR1 function leads to an increase in carotenoids in tomato fruit. PPSR1 affects the abundance of 288 proteins, including phytoene synthase 1 (PSY1), the key rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY1 contains two ubiquitinated lysine residues (Lys380 and Lys406) as revealed by the global analysis and characterization of protein ubiquitination. We provide evidence that PPSR1 interacts with PSY1 precursor protein and mediates its degradation via ubiquitination, thereby affecting the steady-state level of PSY1 protein. Our findings not only uncover a regulatory mechanism for controlling carotenoid biosynthesis, but also provide a strategy for developing carotenoid-enriched horticultural crops.

LIU X X

LI D J

, et al. CRISPR/Cas9-mediated SlAN2 mutants reveal various regulatory models of anthocyanin biosynthesis in tomato plant[J]. Plant cell rep, 2020, 39(6):799-809.

Combining phenotype and gene expression analysis of the CRISPR/Cas9-induced SlAN2 mutants, we revealed that SlAN2 specifically regulated anthocyanin accumulation in vegetative tissues in purple tomato cultivar 'Indigo Rose.' Anthocyanins play an important role in plant development and also exhibit human health benefits. The tomato genome contains four highly homologous anthocyanin-related R2R3-MYB transcription factors: SlAN2, SlANT1, SlANT1-like, and SlAN2-like/Aft. SlAN2-like/Aft regulates anthocyanin accumulation in the fruit; however, the genetic function of the other three factors remains unclear. To better understand the function of R2R3-MYB transcription factors, we conducted targeted mutagenesis of SlAN2 in the purple tomato cultivar 'Indigo Rose' using clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The SlAN2 mutants had a fruit color and anthocyanin content similar to cv. 'Indigo Rose,' while the anthocyanin content and the relative expression levels of several anthocyanin-related genes in vegetative tissues were significantly lower in the SlAN2 mutant relative to cv. Indigo Rose. Furthermore, we found that anthocyanin biosynthesis is controlled by different regulators between tomato hypocotyls and cotyledons. In addition, SlAN2 mutants were shorter, with smaller and lighter fruits than cv. 'Indigo Rose.' Our findings further our understanding of anthocyanin production in tomato and other plant species.

WANG C

WIN J

, et al. Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion[J]. Sci rep, 2017, 7(1):482.

Genome editing has emerged as a technology with a potential to revolutionize plant breeding. In this study, we report on generating, in less than ten months, Tomelo, a non- transgenic tomato variety resistant to the powdery mildew fungal pathogen using the CRISPR/ Cas9 technology. We used wholegenome sequencing to show that Tomelo does not carry any foreign DNA sequences but only carries a deletion that is indistinguishable from naturally occurring mutations. We also present evidence for CRISPR/ Cas9 being a highly precise tool, as we did not detect off- target mutations in Tomelo. Using our pipeline, mutations can be readily introduced into elite or locally adapted tomato varieties in less than a year with relatively minimal effort and investment.

SHELAKE R M

PARK J

, et al. CRISPR/Cas9-mediated generation of pathogen-resistant tomato against tomato yellow leaf curl virus and powdery mildew[J]. Int j mol sci, 2021, 22(4):1878.

YANG X P

YU Y

, et al. Domestication of wild tomato is accelerated by genome editing[J]. Nat biotechnol, 2018, 36:1160-1163.

Crop improvement by inbreeding often results in fitness penalties and loss of genetic diversity. We introduced desirable traits into four stress-tolerant wild-tomato accessions by using multiplex CRISPR-Cas9 editing of coding sequences, cis-regulatory regions or upstream open reading frames of genes associated with morphology, flower and fruit production, and ascorbic acid synthesis. Cas9-free progeny of edited plants had domesticated phenotypes yet retained parental disease resistance and salt tolerance.

ALI Z

ALJEDAANI F

, et al. Engineering resistance against tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato[J]. Plant signal behav, 2018, 13(10):e1525996.

GIMENEZ-IBANEZ S

LEONHARDT N

, et al. Design of a bacterial speck resistant tomato by CRISPR/Cas9-mediated editing of SlJAZ2[J]. Plant biotechnol j, 2019, 17(3):665-673.

Due to their different lifestyles, effective defence against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Solving this trade-off is a major challenge for obtaining broad-spectrum resistance in crops and requires uncoupling the antagonism between the jasmonate (JA) and salicylate (SA) defence pathways. Pseudomonas syringae pv. tomato (Pto) DC3000, the causal agent of tomato bacterial speck disease, produces coronatine (COR) that stimulates stomata opening and facilitates bacterial leaf colonization. In Arabidopsis, stomata response to COR requires the COR co-receptor AtJAZ2, and dominant AtJAZ2Δjas repressors resistant to proteasomal degradation prevent stomatal opening by COR. Here, we report the generation of a tomato variety resistant to the bacterial speck disease caused by PtoDC3000 without compromising resistance to necrotrophs. We identified the functional ortholog of AtJAZ2 in tomato, found that preferentially accumulates in stomata and proved that SlJAZ2 is a major co-receptor of COR in stomatal guard cells. SlJAZ2 was edited using CRISPR/Cas9 to generate dominant JAZ2 repressors lacking the C-terminal Jas domain (SlJAZ2Δjas). SlJAZ2Δjas prevented stomatal reopening by COR and provided resistance to PtoDC3000. Water transpiration rate and resistance to the necrotrophic fungal pathogen Botrytis cinerea, causal agent of the tomato gray mold, remained unaltered in Sljaz2Δjas plants. Our results solve the defence trade-off in a crop, by spatially uncoupling the SA-JA hormonal antagonism at the stomata, entry gates of specific microbes such as PtoDC3000. Moreover, our results also constitute a novel CRISPR/Cas-based strategy for crop protection that could be readily implemented in the field.© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

PERROT L

CHAUVIN L

, et al. Transgene-free genome editing in tomato and potato plants using Agrobacterium-mediated delivery of a CRISPR/Cas9 cytidine base editor[J]. Int j mol sci, 2019, 20(2):402.

NASSAR J A

KHEREDIN S M

, et al. CRISPR/Cas9-mediated mutagenesis of CAROTENOID CLEAVAGE DIOXYGENASE 8 in tomato provides resistance against the parasitic weed Phelipanche aegyptiaca[J]. Sci rep, 2019, 9(1):11438.

JAYASINGHE W H

KWON J

, et al. Artificially edited alleles of the eukaryotic translation initiation factor 4e1 gene differentially reduce susceptibility to cucumber mosaic virus and potato virus y in tomato[J]. Front microbiol, 2020, 11:564310.

VENKATESH J

LEE J H

, et al. Genome editing of eIF4E1 in tomato confers resistance to pepper mottle virus[J]. Front plant sci, 2020, 11:1098.

LIU C X

ZHAO R R

, et al. CRISPR/Cas9-Mediated SlNPR1 mutagenesis reduces tomato plant drought tolerance[J]. BMC plant biol, 2019, 19(1):38.

NPR1, nonexpressor of pathogenesis-related gene 1, is a master regulator involved in plant defense response to pathogens, and its regulatory mechanism in the defense pathway has been relatively clear. However, information about the function of NPR1 in plant response to abiotic stress is still limited. Tomato is the fourth most economically crop worldwide and also one of the best-characterized model plants employed in genetic studies. Because of the lack of a stable tomato NPR1 (SlNPR1) mutant, little is known about the function of SlNPR1 in tomato response to biotic and abiotic stresses.Here we isolated SlNPR1 from tomato 'Ailsa Craig' and generated slnpr1 mutants using the CRISPR/Cas9 system. Analysis of the cis-acting elements indicated that SlNPR1 might be involved in tomato plant response to drought stress. Expression pattern analysis showed that SlNPR1 was expressed in all plant tissues, and it was strongly induced by drought stress. Thus, we investigated the function of SlNPR1 in tomato-plant drought tolerance. Results showed that slnpr1 mutants exhibited reduced drought tolerance with increased stomatal aperture, higher electrolytic leakage, malondialdehyde (MDA) and hydrogen peroxide (HO) levels, and lower activity levels of antioxidant enzymes, compared to wild type (WT) plants. The reduced drought tolerance of slnpr1 mutants was further reflected by the down-regulated expression of drought related key genes, including SlGST, SlDHN, and SlDREB.Collectively, the data suggest that SlNPR1 is involved in regulating tomato plant drought response. These results aid in further understanding the molecular basis underlying SlNPR1 mediation of tomato drought sensitivity.

RAHIM M A

JUNG H J

, et al. Genome-wide characterization of NBS-encoding genes in watermelon and their potential association with gummy stem blight resistance[J]. Int j mol sci, 2019, 20(4):902.

STASKAWICZ B

WEIGEL D

, et al. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease[J]. Nat biotechnol, 2013, 31:691-693.

JIANG L J

GAO Q

, et al. Efficient CRISPR/Cas9-based gene knockout in watermelon[J]. Plant cell rep, 2017, 36(3):399-406.

CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

CHANG J J

LI J Y

, et al. Disruption of the bHLH transcription factor abnormal tapetum 1 causes male sterility in watermelon[J]. Hortic res, 2021, 8(1):258.

WANG J F

GUO S G

, et al. CRISPR/Cas9-mediated mutagenesis of ClBG1 decreased seed size and promoted seed germination in watermelon[J]. Hortic res, 2021, 8(1):70.

ZHAO X Q

LING K S

, et al. The FonSIX6 gene acts as an avirulence effector in the Fusarium oxysporum f. sp. niveum - watermelon pathosystem[J]. Sci rep, 2016, 6:28146.

LIU Q L

YANG X P

, et al. CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum[J]. Plant cell rep, 2020, 39(5):589-595.

JIANG L J

CUI X X

, et al. Engineering herbicide-resistant watermelon variety through CRISPR/Cas9-mediated base-editing[J]. Plant cell rep, 2018, 37(9):1353-1356.

ZHAO S J

SUN H H

, et al. Resequencing of 414 cultivated and wild watermelon accessions identifies selection for fruit quality traits[J]. Nat genet, 2019, 51(11):1616-1623.

Fruit characteristics of sweet watermelon are largely the result of human selection. Here we report an improved watermelon reference genome and whole-genome resequencing of 414 accessions representing all extant species in the Citrullus genus. Population genomic analyses reveal the evolutionary history of Citrullus, suggesting independent evolutions in Citrullus amarus and the lineage containing Citrullus lanatus and Citrullus mucosospermus. Our findings indicate that different loci affecting watermelon fruit size have been under selection during speciation, domestication and improvement. A non-bitter allele, arising in the progenitor of sweet watermelon, is largely fixed in C. lanatus. Selection for flesh sweetness started in the progenitor of C. lanatus and continues through modern breeding on loci controlling raffinose catabolism and sugar transport. Fruit flesh coloration and sugar accumulation might have co-evolved through shared genetic components including a sugar transporter gene. This study provides valuable genomic resources and sheds light on watermelon speciation and breeding history.

LI M Y

GUO S G

, et al. Evolutionary gain of oligosaccharide hydrolysis and sugar transport enhanced carbohydrate partitioning in sweet watermelon fruits[J]. Plant cell, 2021, 33(5):1554-1573.

GUO S G

JI G J

, et al. A unique chromosome translocation disrupting ClWIP1 leads to gynoecy in watermelon[J]. Plant J, 2020, 101(2):265-277.

GUO Y L

YAN J Y

, et al. The role of watermelon caffeic acid O-methyltransferase (ClCOMT1) in melatonin biosynthesis and abiotic stress tolerance[J]. Hortic res, 2021, 8(1):210.

SHORINOLA O

STACEY N

, et al. Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease[J]. Genome biol, 2015, 16:258.

ZHU C Z

ZHENG M

, et al. CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. Capitata using the endogenous tRNA-processing system[J]. Hortic res, 2019, 6:20.

ROBIN A H

YANG K

, et al. Developmental and genotypic variation in leaf wax content and composition, and in expression of wax biosynthetic genes in Brassica oleracea var. capitata[J]. Front plant sci, 2017, 7:1972.

TANG J

LIU Z M

, et al. Cgl2 plays an essential role in cuticular wax biosynthesis in cabbage (Brassica oleracea var. Capitata)[J]. BMC plant biol, 2017, 17(1):223.

DONG X

JI J L

, et al. BoCER1 is essential for the synthesis of cuticular wax in cabbage (Brassica oleracea var. Capitata)[J]. Sci hortic, 2021, 277:109801.

JIANG M

ZHENG H

, et al. Color-related chlorophyll and carotenoid concentrations of Chinese kale can be altered through CRISPR/Cas9 targeted editing of the carotenoid isomerase gene BoaCRTISO[J]. Hortic res, 2020, 7(1):161.

OLESZKIEWICZ T

LOWDER L G

, et al. Efficient CRISPR/Cas9-based genome editing in carrot cells[J]. Plant cell rep, 2018, 37(4):575-586.

FENG K

XIONG A S

. CRISPR/Cas9-mediated multiply targeted mutagenesis in orange and purple carrot plants[J]. Mol biotechnol, 2019, 61(3):191-199.

BRUMIN M

WOLF D

, et al. Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology[J]. Mol plant pathol, 2016, 17(7):1140-1153.

Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N' and C' termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.© 2016 BSPP and John Wiley & Sons Ltd.

LI D W

LIU X

, et al. Engineering non-transgenic gynoecious cucumber using an improved transformation protocol and optimized CRISPR/Cas9 System[J]. Mol plant, 2017, 10(12):1575-1578.

ZHANG Z

LI S

, et al. Genetic regulation of ethylene dosage for cucumber fruit elongation[J]. Plant cell, 2019, 31(5):1063-1076.

Plant organ growth and development are determined by a subtle balance between growth stimulation and inhibition. Fruit size and shape are important quality traits influencing yield and market value; however, the underlying mechanism regulating the balance of fruit growth to achieve final size and shape is not well understood. Here, we report a mechanistic model that governs cucumber (Cucumis sativus) fruit elongation through fine-tuning of ethylene homeostasis. We identified a cucumber mutant that bears short fruits owing to repressed cell division. SF1 (Short Fruit 1) encodes a cucurbit-specific RING-type E3 ligase, and the mutation resulted in its enhanced self-ubiquitination and degradation, but accumulation of ACS2 (1-aminocyclopropane-1-carboxylate synthase 2), a rate-limiting enzyme for ethylene biosynthesis. The overproduction of ethylene contributes to the short-fruit phenotype of sf1. Dysfunction of ACS2 resulted in reduced ethylene production, but still repressed cell division and shorter fruit, suggesting that ethylene is still required for basal fruit elongation. SF1 ubiquitinates and degrades both itself and ACS2 to control ethylene synthesis for dose-dependent effect on cell division and fruit elongation. Our findings reveal the mechanism by which ethylene dosage is regulated for the control of cell division in developing fruit.

YAN J B

ZHANG Z

, et al. Regulation of plant architecture by a new histone acetyltransferase targeting gene bodies[J]. Nat plants, 2020, 6(7):809-822.

Axillary meristem development determines both plant architecture and crop yield; this critical process is regulated by the PROLIFERATING CELL FACTORS (TCP) family of transcription factors. Although TCP proteins bind primarily to promoter regions, some also target gene bodies for expression activation. However, the underlying regulatory mechanism remains unknown. Here we show that TEN, a TCP from cucumber (Cucumis sativus L.), controls the identity and mobility of tendrils. Through its C terminus, TEN binds at intragenic enhancers of target genes; its N-terminal domain functions as a non-canonical histone acetyltransferase (HAT) to preferentially act on lysine 56 and 122 of the histone H3 globular domain. This HAT activity is responsible for chromatin loosening and host-gene activation. The N termini of all tested CYCLOIDEA and TEOSINTE BRANCHED 1-like TCP proteins contain an intrinsically disordered region; despite their sequence divergence, they have conserved HAT activity. This study identifies a non-canonical class of HATs and provides a mechanism by which modification at the H3 globular domain is integrated with the transcription process.

WANG B

WANG S

, et al. Genome-wide target mapping shows histone deacetylase complex1 regulates cell proliferation in cucumber fruit[J]. Plant physiol, 2020, 182(1):167-184.

Histone deacetylase (HDAC) proteins participate in diverse and tissue-specific developmental processes by forming various corepressor complexes with different regulatory subunits. An important HDAC machinery hub, the Histone Deacetylase Complex1 (HDC1) protein, participates in multiple protein-protein interactions and regulates organ size in plants. However, the mechanistic basis for this regulation remains unclear. Here, we identified a cucumber () short-fruit mutant () with a phenotype that includes repressed cell proliferation. encodes an HDC1 homolog, and its expression is enriched in meristematic tissues, consistent with a role in regulating cell proliferation through the HDAC complex. A weak allele impairs HDAC targeting to chromatin, resulting in elevated levels of histone acetylation. Genome-wide mapping revealed that SF2 directly targets and promotes histone deacetylation associated with key genes involved in multiple phytohormone pathways and cell cycle regulation, by either directly repressing or activating their expression. We further show that SF2 controls fruit cell proliferation through targeting the biosynthesis and metabolism of cytokinin and polyamines. Our findings reveal a complex regulatory network of fruit cell proliferation mediated by HDC1 and elucidate patterns of HDC1-mediated regulation of gene expression.© 2020 American Society of Plant Biologists. All Rights Reserved.

WANG L M

HAN L J

, et al. HECATE2 acts with GLABROUS3 and Tu to boost cytokinin biosynthesis and regulate cucumber fruit wart formation[J]. Plant physiol, 2021, 187(3):1619-1635.

Warty fruit in cucumber (Cucumis sativus L.) is an important quality trait that greatly affects fruit appearance and market value. The cucumber wart consists of fruit trichomes (spines) and underlying tubercules, in which the existence of spines is prerequisite for tubercule formation. Although several regulators have been reported to mediate spine or tubercule formation, the direct link between spine and tubercule development remains unknown. Here, we found that the basic Helix-Loop-Helix (bHLH) gene HECATE2 (CsHEC2) was highly expressed in cucumber fruit peels including spines and tubercules. Knockout of CsHEC2 by the CRISPR/Cas9 system resulted in reduced wart density and decreased cytokinin (CTK) accumulation in the fruit peel, whereas overexpression of CsHEC2 led to elevated wart density and CTK level. CsHEC2 is directly bound to the promoter of the CTK hydroxylase-like1 gene (CsCHL1) that catalyzes CTK biosynthesis, and activated CsCHL1 expression. Moreover, CsHEC2 physically interacted with GLABROUS3 (CsGL3, a key spine regulator) and Tuberculate fruit (CsTu, a core tubercule formation factor), and such interactions further enhanced CsHEC2-mediated CsCHL1 expression. These data suggested that CsHEC2 promotes wart formation by acting as an important cofactor for CsGL3 and CsTu to directly stimulate CTK biosynthesis in cucumber. Thus, CsHEC2 can serve as a valuable target for molecular breeding of cucumber varieties with different wart density requirements.© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.

LI S

YANG L

, et al. Gain-of-function of the 1-aminocyclopropane-1-carboxylate synthase gene ACS1G induces female flower development in cucumber gynoecy[J]. Plant cell, 2021, 33(2):306-321.

TURESSON H

NICOLIA A

, et al. Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts[J]. Plant cell rep, 2017, 36(1):117-128.

Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. Site-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2-12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1-10 bp, but also vector DNA inserts of 34-236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.

AKIYAMA R

LEE H J

, et al. Generation of α-solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene[J]. Plant physiol biochem, 2018, 131:70-77.

PENG Z

TANG D

, et al. Generation of self-compatible diploid potato by knockout of S-RNase[J]. Nat plants, 2018, 4(9):651-654.

Re-domestication of potato into an inbred line-based diploid crop propagated by seed represents a promising alternative to traditional clonal propagation of tetraploid potato, but self-incompatibility has hindered the development of inbred lines. To address this problem, we created self-compatible diploid potatoes by knocking out the self-incompatibility gene S-RNase using the CRISPR-Cas9 system. This strategy opens new avenues for diploid potato breeding and will also be useful for studying other self-incompatible crops.

ATKINS P A

VOYTAS D F

, et al. Generation and inheritance of targeted mutations in potato (Solanum tuberosum L.) using the CRISPR/Cas system[J]. Plos one, 2015, 10:e0144591.

ZHANG F J

ZHONG Z Y

, et al. Generation of virus-resistant potato plants by RNA genome targeting[J]. Plant biotechnol j, 2019, 17(9):1814-1822.

CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA-targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

LEE A

JO S H

, et al. Nitrogen signaling genes and SOC1 determine the flowering time in a reciprocal negative feedback loop in Chinese cabbage (Brassica rapa L.) based on CRISPR/Cas9-mediated mutagenesis of multiple BrSOC1 homologs[J]. Int j mol sci, 2021, 22(9):4631.

AHN H

RYU J

, et al. Generation of early-flowering chinese cabbage (Brassica rapa spp. pekinensis ) through Crispr/Cas9-mediated genome editing[J]. Plant biotechnol rep, 2019, 13(5):491-499.

GAGNEUL D

ALVES DOS SANTOS H

, et al. Efficient genome editing using CRISPR/Cas9 technology in chicory[J]. Int j mol sci, 2019, 20:1155.

RON M

HUO H

, et al. High-resolution analysis of the efficiency, heritability, and editing outcomes of CRISPR/Cas9-induced modifications of NCED4 in lettuce (Lactuca sativa)[J]. G3 (Bethesda), 2018, 8(5):1513-1521.

LÓPEZ-CRISTOFFANINI C

NOGUÉS S

. Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon[J]. Sci rep, 2019, 9(1):17077.

CRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42-45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.

TIAN H J

MA Y L

, et al. Targeted creating new mutants with compact plant architecture using CRISPR/Cas9 genome editing by an optimized genetic transformation procedure in cucurbit plants[J]. Hortic res, 2022, uhab086.

CAO H S

YANG L

, et al. Tissue-specific respiratory burst oxidase homolog-dependent H2O2 signaling to the plasma membrane H+-ATPase confers potassium uptake and salinity tolerance in Cucurbitaceae[J]. J exp bot, 2019, 70(20):5879-5893.

SOHAIL H Z

CAO H S

, et al. An efficient root transformation system for CRISPR/Cas9-based analyses of shoot-root communication in cucurbit crops[J]. Hortic res, 2022, 20:uhab082.

GIANOGLIO S

MOGLIA A

, et al. Simultaneous CRISPR/Cas9 editing of three PPO genes reduces fruit flesh browning in Solanum melongena L.[J]. Front plant sci, 2020, 11:607161.

CHOI J

WON K H

. A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum[J]. BMC plant bio, 2020, l20(1):449.

KIM H B

JEON H J

, et al. Agrobacterium-mediated Capsicum annuum gene editing in two cultivars, hot pepper CM334 and bell pepper dempsey[J]. Int j mol sci, 2021, 22(8):3921.

MOHANTY J N

MAHANTY B

, et al. A single transcript CRISPR/Cas9 mediated mutagenesis of CaERF28 confers anthracnose resistance in chilli pepper (Capsicum annuum L.)[J]. Planta, 2021, 254(1):5.

MASSEL K

GODWIN I D

, et al. Applications and potential of genome editing in crop improvement[J]. Genome biol, 2018, 19(1):210.

Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism's genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.

YANG F

ZHANG J J

, et al. Application of CRISPR/Cas9 in crop quality improvement[J]. Int j mol sci, 2021, 22(8):4206.

SUN H H

GUO S G

, et al. Decreased protein abundance of lycopene β-cyclase contributes to red flesh in domesticated watermelon[J]. Plant physiol, 2020b, 183(3):1171-1183.

PRIBIL M

PALMGREN M

, et al. A CRISPR way for accelerating improvement of food crops[J]. Nat food, 2020, 1:200-205.

LA RUSSA M

QI L S

. CRISPR/Cas9 in genome editing and beyond[J]. Annu rev biochem, 2016, 85:227-264.

The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

ABDULAH SNA

. CRISPR/dCas9 platforms in plants:strategies and applications beyond genome editing[J]. Plant biotechnol j, 2019, 18:32-44.

ZHOU P

YUEN C T L

, et al. Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9[J]. Nat methods, 2019, 16(8):722-730.

The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.

JOSEPHS E A

BHANDARKAR V

, et al. Increasing the specificity of CRISPR systems with engineered RNA secondary structures[J]. Nat biotechnol, 2019, 37:657-666.

CRISPR (clustered regularly interspaced short palindromic repeat) systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcus pyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems.

YADAV N S

OROZCO E M J

, et al. Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts[J]. Peerj, 2020, 8:e8362.

BAE S J

LEE S

, et al. Chloroplast and mitochondrial DNA editing in plants[J]. Nat plants, 2021, 7(7):899-905.

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