Gene editing: an instrument for practical application of gene biology to plant breeding

Gene editing: an instrument for practical application of gene biology to plant breeding 基因编辑:将基因生物学用于植物育种的工具 1 2 first-author 1 1 1 1 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:qyshu@zju.edu.cn †E-mail:qyshu@zju.edu.cn qyshu@zju.edu.cn National Key Laboratory of Rice Biology, Institute of Crop Sciences, Zhejiang University, Hangzhou 310058, China National Key Laboratory of Rice Biology, Institute of Crop Sciences, Zhejiang University, Hangzhou 310058, China The Rural Development Academy, Zhejiang University, Hangzhou 310058, China The Rural Development Academy, Zhejiang University, Hangzhou 310058, China Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by both new knowledge on trait development and regulation (e.g., functional genomics) and new technologies (e.g., biotechnologies and phenomics). Gene editing, particularly by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and its variants, has become a powerful technology in plant research and may become a game-changer in plant breeding. Traits are conferred by coding and non-coding genes. From this perspective, we propose different editing strategies for these two types of genes. The activity of an encoded enzyme and its quantity are regulated at transcriptional and post-transcriptional, as well as translational and post-translational, levels. Different strategies are proposed to intervene to generate gene functional variations and consequently phenotype changes. For non-coding genes, trait modification could be achieved by regulating transcription of their own or target genes via gene editing. Also included is a scheme of protoplast editing to make gene editing more applicable in plant breeding. In summary, this review provides breeders with a host of options to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding. 人口不断增长给世界粮食安全带来了严峻的挑战,植物育种是应对这一挑战的最重要手段之一.过去三十年来,性状形成和调控的新知识(如功能基因组学)和新技术(如生物信息学和表型组学)极大地支持了植物育种的发展.基因编辑,特别是基于CRISPR/Cas技术和其衍生技术,已成为强有力的植物研究技术,可能直接改变植物育种的方法和策略.植物表型性状受编码基因和非编码基因的控制,在本文中,我们提出了编辑这两类基因的不同策略.对于编码基因,其编码蛋白的活性和数量可在转录和转录后水平以及翻译和翻译后水平加以调节,我们由此提出了创造基因功能性变异从而改变性状表型的基因编辑策略.对于非编码基因,则可以采用基因编辑技术对其转录水平或对靶基因的目标序列加以改造,达到产生新的性状的目的.此外,我们还提出了一种基于原生质体的基因编辑方案,使基因编辑技术更适合于植物育种.总之,本文提出了一系列可供植物育种者选择的将基因生物学知识转化为实用育种策略的方案,即基因编辑技术成为将基因生物学知识用于植物育种的技术. Gene editing Expression regulation Novel allele Trait development Plant breeding 基因编辑 表达调控 新等位基因 性状形成 植物育种 Introduction 1 Plant breeding is widely expected to help meet the challenge of feeding a population of 10 billion people on earth (Hickey et al., 2019 2019 ). The fulfillment of this magnificent task will rely on advances and breakthroughs in all fields related to plant breeding, including new techniques yet to be innovated. Gene editing has recently emerged as such a breakthrough technology. It was not even dreamed of ten years ago in this field. Gene editing started with zinc-finger nucleases (ZFNs) (Lloyd et al., 2005 2005 ) and later, transcription activator-like effector nucleases (TALENs) (Cermak et al., 2011 2011 ; Mahfouz et al., 2011 2011 ). It is now dominated by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology (Li et al., 2013 2013 ; Nekrasov et al., 2013 2013 ; Shan et al., 2013 2013 ). Although the CRISPR/Cas technology has been in being for only seven years, its development as a new technology has been unprecedented. Many CRISPR variant technologies have already been developed and quickly deployed for basic and applied research, including plant breeding (Li et al., 2013 2013 ; Chavez et al., 2015 2015 ; Piatek et al., 2015 2015 ; Xie et al., 2015 2015 ; Zetsche et al., 2015 2015 ; Endo et al., 2016 2016 ; Kleinstiver et al., 2016 2016 ; Lin et al., 2016 2016 ; Minkenberg et al., 2017 2017 ; Shimatani et al., 2017 2017 ; Tang L et al., 2017 2017 ; Hu et al., 2018 2018 ; Nishimasu et al., 2018 2018 ; Ren et al., 2019 2019 ). The progress on and perspectives of genomic editing in plant breeding have already been extensively reviewed recently (Zhang H et al., 2017 2017 ; Pandiarajan and Grover, 2018 2018 ; Zaidi et al., 2018 2018 ; Eş et al., 2019 2019 ; Hua et al., 2019 2019 ; Mao et al., 2019 2019 ; Molla and Yang, 2019 2019 ; Kausch et al., 2019 2019 ; Zhang YX et al., 2019 2019 ; Zimny et al., 2019 2019 ). Some reviews were published almost simultaneously but in different journals, and hence the key information, insights, and perspectives conveyed in these reviews overlapped to some extent. These reviews mainly covered topics such as: (1) the ever-developing gene editing technologies adapted to plant systems; (2) the applications of gene editing in plant breeding for different breeding objectives; (3) challenges and perspectives of gene editing in plant breeding. In summary, it is now well recognized that gene editing has a broad spectrum of applications, from knocking out a gene to quickly generating a recessive but desired trait (such as fragrance, early maturity) (Shan et al., 2015 2015 ; Xu et al., 2016 2016 ; Soyk et al., 2017 2017 ), to generating new alleles by nucleotide substitution to create a novel trait (Li et al., 2016 2016 ; Hua et al., 2018 2018 ; Zong et al., 2018 2018 ), to quantitatively regulating the expression of a particular gene to increase stress tolerance or content of desired metabolites (Li et al., 2012 2012 ; Čermák et al., 2015 2015 ; Jia et al., 2017 2017 ), to name a few. In parallel with rapid technical advance in plant research, study on the mechanism of how a gene exerts its function and how a gene is regulated, i.e., gene biology, has also experienced tremendous progress in the past two decades. However, due to the lack/incomprehension of such knowledge, and in most cases lack of proper techniques to make use of, plant breeders at large have not taken advantage of this knowledge in their breeding programs. Although some of the mechanisms have already been integrated into gene editing for generation of novel traits, a comprehensive examination of how to make use of gene biology in gene editing-facilitated plant breeding has been lacking. In this perspective paper, we review the current state of plant gene biology research, from gene structure to transcription and post-transcription regulations, to translation and post-translation modifications, and propose various gene editing strategies that could be deployed to generate novel alleles of a given gene for a particular purpose in plant breeding. That is, to use gene editing to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding. A brief introduction to gene biology: type of genes, functioning mechanisms, and regulation at various levels 1 Our understanding of a gene and how it functions has evolved drastically since the term “gene” was first introduced into biology. Nowadays, genes are divided into two basic types: coding and non-coding. Proteins encoded by coding genes also vary greatly in their function and can be divided into different categories: (1) enzymes involved in biochemical catalytic reactions in metabolism (classical enzymes) and (2) enzymes specialized in modification of proteins, RNAs, and DNAs; (3) non-enzyme proteins such as transporters of ions and macromolecules and (4) non-enzyme proteins working together with other proteins in biochemical process or gene regulation, to name a few. Non-coding genes can also be further classified into long non-coding genes and small RNA genes. Genes of different types function in distinct ways to confer a phenotype. Consequently, different strategies could be developed to manipulate their function to achieve a specific objective in plant breeding. For a coding gene to confer a phenotype in plants, it should first be transcribed into RNA, processed into messenger RNA (mRNA), and translated into a peptide, which should be modified before becoming active, or be transported into proper organelles for functioning. Therefore, the function of a coding gene could be regulated at transcriptional, post-transcriptional, and translational levels, and further modified post-translationally. For non-coding genes, their products are often involved in regulation of other genes through distinct or as yet unknown mechanisms, and hence their function could also be regulated or modified. In the following sections, the biological basis of gene expression and regulation is elaborated in more detail with practical examples when available, and respective ways to modify traits by gene editing are proposed for plant breeding. Gene editing for transcription and post-transcription regulations 1 Transcription is a process in which DNA is transcribed into RNA, which involves three steps, i.e., initiation, elongation, and termination. Transcription begins with the binding of RNA polymerase to DNA at the promoter region, and RNA polymerase works together with transcriptional factor (TF) in plants. The TFs can either bind with the RNA polymerase to form complexes or directly bind with the specific sequence on the promoter region. Therefore, the transcription of a gene of interest (GOI) could be altered by editing either its sequence (e.g., promoter, terminator) or its TFs. Transcribed RNA is processed into mRNA for translation, and the stability of mRNA is dependent on sequences in the untranslated regions (UTRs) as well as others. Editing for transcription 2 Functionally, the promoter region consists of three important components, i.e., the core/minimal, distal, and proximal promoter regions. The core/minimal promoter where the TATA box, initiator, and B recognition element (BRE) reside is the sequence that is necessary for initiating transcription. The distal promoter contains enhancers or silencers and can also influence the expression of a gene. The proximal promoter region has several TF-binding sites (TFBSs) adjacent to the core promoter sequence (Fig. 1 1 ). Cis- regulatory elements (CREs) are known to be one or more TFBSs in the proximal/distal promoter region (Pandiarajan and Grover, 2018 2018 ). CRE: Cis regulatory element; CRISPR: clustered regularly interspaced short palindromic repeat; Cas: CRISPR-associated protein In the promoter region, many different types of responsive elements have been reported that can induce transcription when stimulated by various biotic or abiotic stress, such as a responsive element to oxidative stress, environmental stress (light response elements (LREs), heat-shock element (HSE)), or pathogen (HSR203 responsive element (HSRE)) and responsive elements to different hormones (such as abscisic acid (ABA)-responsive element (ABRE) and gibberellin (GA)-responsive element (GARE)). The transcription proceeds for the elongation with the RNA polymerase after initiation and the template DNA strand is transcribed into RNA. At termination, RNA polymerase releases from DNA and the transcription stops. Therefore, gene transcription can be regulated by inhibiting the initial binding of the promoter with the RNA polymerase and TFs. This can be achieved by targeting the known vital elements on the promoter or unknown elements analyzed by the websites through the CRISPR/Cas technology. Many important traits have evolved from sequence changes in the promoter region during the course of crop domestication, e.g., grain color (Espley et al., 2009 2009 ; Butelli et al., 2012 2012 ; Oikawa et al., 2015 2015 ) and shape (Li et al., 2011 2011 ; Wang et al., 2012 2012 , 2015 2015 ; Xu et al., 2015 2015 ), and resistance to disease (Li et al., 2017 2017 ). They often resulted from altered expression levels of genes responsive to hormonal or environmental signals. In practice, it has been proven that genome editing can be used to change gene transcription by editing CREs of the GOI (gene B in Fig. 1 1 ), or by knocking out or altering transcription of its TFs (gene A in Fig. 1 1 ), thus improving stress tolerance, fruit size, and disease resistance in crops, such as rice, wheat, and tomato (Wang YP et al., 2014 2014 ; Wang FJ et al., 2016 2016 ; Rodriguez-Leal et al., 2017 2017 ; Shi et al., 2017 2017 ). Editing of UTR 2 The UTR region contains the sequences ahead of the translation initiation codon ATG (5'-UTR) and after the stop codon (3'-UTR) in mature mRNA. 5'-UTR works as the entry point for the ribosome during translation of mRNA and contains elements that can interact with thermal sensors and other classes of translation or transcription attenuators, and hence plays crucial roles in the regulation of gene transcription through influencing the mRNA stability in response to environmental stimuli (Leppek et al., 2018 2018 ; Al-Zeer et al., 2019 2019 ). Therefore, the transcription of a GOI could theoretically be altered by targeted mutation in UTRs. Editing for post-transcriptional regulation 2 Many genes are subject to post-transcriptional regulation in plants, e.g., alternative splicing (AS), alternative polyadenylation (APA), and microRNA (miRNA) digestion (Fig. 2 2 ), which affects transcript processing and protein translation (Deng and Cao, 2017 2017 ). CRISPR: clustered regularly interspaced short palindromic repeat; Cas: CRISPR-associated protein; miRNA: microRNA; mRNA: messenger RNA; UTR: untranslated region AS rearranges the pattern of intron and exon elements in mRNAs to direct the decay of a special RNA variant. This enables a gene to encode diverse protein isoforms (Filichkin et al., 2015 2015 ). For example, one of the florigen genes in temperate grasses is capable of producing two splicing isoforms that play antagonistic roles in flowering control, suggestive of a pervasive phenomenon by AS to confer a gene with different cellular functions or properties (Qin et al., 2017 2017 ). Most eukaryotic splicing processes of pre-mRNAs observe a GU/AG rule (Reddy et al., 2013 2013 ), and thus it can be modified by a CRISPR/Cas9 technology. The recently developed Cas9-directed base editor, which can introduce desired point mutation, has been shown to be an ideally efficient way to manipulate splicing outcomes. For example, hypersensitive to ABA1 ( HAB1 ), a phosphatase 2C gene in Arabidopsis, that can generate two isoforms playing entirely opposite roles in the GA signaling, has successfully produced a retention of intron (Xue et al., 2018 2018 ). The ABA hypersensitive phenotype of transgenic mutants demonstrated that generation of HAB1 AS isoforms had been completely prevented through single-base substitutions (Xue et al., 2018 2018 ). As the base editing tools with non-specific protospacer adjacent motif (PAM) properties are becoming available, manipulating pre-mRNA splicing via CRISPR-directed base editors will gain wide application in mRNA isoform functional studies in crops. Similar to transcriptional control, the strategy of gene editing, especially by CRISPR/Cas9, has now revealed significant insights into post-transcriptional effects. miRNAs, a class of 20-to 24-nt RNAs generated from a stem-loop structure, play regulatory roles in a wide range of biological processes in eukaryotes (Wu et al., 2010 2010 ). Besides deletion of the full miRNA gene locus, making indels in miRNA precursors by CRISPR/Cas9 could also readily abolish miRNA functions, because the secondary structure of the miRNA precursor is critical for the efficiency of miRNA formation. Generally, plant miRNAs have almost perfect sequence complementarity with targets. On the one hand, it is simple to destroy the regulatory module of miRNA and targets. This is done through the introduction of indels where miRNA and target pairs, because of the matching degree at miRNA and target sites, especially in the 5' end (positions 2 to 8) and central region (positions 10 and 11) of miRNA, are crucial for the efficiency of miRNA-mediated cleavage. On the other hand, it is also possible to artificially fine-tune the target expression through adjustment of complementary miRNA and target sites by precise base editing. In a recent study, Zhang’s group attempted to produce miR528 loss-of-function mutants through CRISPR/Cas9-based genome editing (Zhou et al., 2017 2017 ). The transgenic plants with 3-bp deletion in miR528 precursor were, as expected, observed to compromise miR528 function in salt stress response. However, those with 1-bp insertion in miR528 precursor have no obvious effects. This interesting finding indicates that creating miRNA locus with large deletions is more credible than engineering miRNA precursors to obtain miRNA loss-of-function mutants via CRISPR/Cas9. Another example is to fine-tune plant architecture by miRNA editing. Promotion of high resource use efficiency via ideal plant architecture (IPA) is an attractive way to increase crop yield potential (Jiao et al., 2010 2010 ). Rice SQUAMOSA promoter-binding-like 14 ( OsSPL14 ), an SQUAMOSA promoter-binding protein (SBP)-domain transcription factor targeted by miR156, is the locus of IPA1 and can be exploited to increase grain yield through balancing panicle size and tiller number, if it is optimally expressed (Zhang L et al., 2017 2017 ). Although selection of novel promoter alleles in natural or mutagenized populations has been suggested as a useful way to control the IPA1 expression pattern, genome editing at miR156 and IPA1 target site would be an alternative way to better fine-tune IPA1 to design elite super rice varieties. Like genomic editing, RNA editing is also a mode of nucleotide modification. However, it is usually a natural phenomenon occurring at the RNA rather than DNA nucleotides from their genome-encoded sequence, which is acting as an indirect repair mechanism that corrects DNA mutations at the RNA level (Takenaka et al., 2013 2013 ). In flowering plants, mRNA editing is generally performed as a C-to-U alteration, and subsequently results in a change of protein amino acids or gene expression via AS or miRNA-mediated degradation (Takenaka et al., 2013 2013 ). Since RNA editing is not always emergent, people can directly edit a DNA nucleotide sequence at an RNA editing site by CRISPR/Cas9 to artificially control the physiology caused by the occasional RNA editing event. APA is a widespread gene regulation mechanism that generates mRNA with different 3'-UTRs, allowing them to be differentially degraded or interact with diverse sets of RNA regulators such as RNA-binding proteins and miRNAs (Hunt, 2014 2014 ). Because APA generally occurs at different sites enriched with poly(A), it is conceivable to change APA events through deletion or alternation of poly(A) fragments by the CRISPR/Cas9 method to form truncated mRNA isoforms with different adenylations In theory, gene editing could be used in all aspects of post-transcription regulation; however, there is no report yet on success of APA or RNA editing events so far by CRISPR/Cas genome editing, and even applications of gene editing on miRNA and AS are still very limited. It may well be in that the single nucleotide editing efficiency is not yet high enough. Nevertheless, with the progress made via the breakthroughs of base editing tools, genome editing promises to contribute a lot to manipulating gene expression explicitly at post-transcription levels. Gene editing for translation, post-translation modification, and protein localization 1 Classical gene editing often results in indel mutations in the targeted genome region, and when it occurs in the exonic region, it would completely or partially knock out a protein-coding gene. Indeed, many important traits, selected by farmers during the domestication process of various crops, are controlled by recessive alleles, such as the “green revolution” gene semi-dwarf 1 ( sd1 ), the fragrance gene betaine aldehyde dehydrogenase 2 ( badh2 ), and the long grain gene grain size 3 ( gs3 ) in rice. Hence, gene editing has already been successfully applied to quickly breed a trait that is in demand, such as fragrance, waxy rice, low phytic acid, and low Cd accumulation (Shan et al., 2015 2015 ; Sun et al., 2017 2017 ; Tang X et al., 2017 2017 ; Zhang JS et al., 2018 2018 ; Zhou et al., 2018 2018 ; Jiang et al., 2019 2019 ; Liu et al., 2019 2019 ). By using base editing techniques, the amino acid sequence could also be modified to produce partial knockout or gain-of-function mutations through various mechanisms (Fig. 3 3 ). (a) Frameshift mutations cause loss-of-function alleles; (b) Amino acid substitutions cause partial loss-of-function or gain-of-function alleles. DSB: double-strand break; UTR: untranslated region Upstream ORF editing for regulating of primary ORF translation 2 It is now considered as a general mechanism that upstream ORFs (uORFs) control the amount of protein that is synthesized from their downstream primary ORFs (pORFs). According to von Arnim et al. ( 2014 2014 ), from about 30% to more than 40% transcripts harbor uORFs in plants, including important crop plants such as maize and rice. By demonstrating that genome editing of endogenous uORFs could enable the modulation of translation of mRNAs from four pORFs that are involved in either development or antioxidant biosynthesis, Zhang HW et al. ( 2018 2018 ) proposed that gene editing could be a generalized instrument for translation regulation in plants. Editing for protein activation and degradation 2 After being transcribed and translated, the complexity of genetic information encoded in DNA is largely increased by multiple post-translational modifications (PTMs) (Fig. 4 4 ). PTMs are chemical alterations to protein structure, typically catalyzed by substrate-specific enzymes, which themselves are under strict control by PTMs. Due to a lot of types of PTMs, there is a rich diversity of gene products (Deribe et al., 2010 2010 ). PTMs might induce conformational changes or form a docking site to mediate molecular recognition and stabilize protein–ligand and protein–protein interactions. By reversible multisite PTMs, they can rapidly and dynamically regulate protein turnover, localization, and activation, and thus dynamically coordinate developmental processes (Huber and Hardin, 2004 2004 ; Ytterberg and Jensen, 2010 2010 ; Duan and Walther, 2015 2015 ). PTMs play the key role in plant development stages. For example, GENOMES UNCOUPLED 4 (GUN4) is a positive regulator of light-dependent chlorophyll biosynthesis. GUN4 activates Mg chelatase (MgCh) that catalyzes the insertion of an Mg 2+ ion into protoporphyrin IX. Arabidopsis thaliana GUN4 is phosphorylated at Ser264 (S264), the penultimate amino acid residue at the C terminus. Phosphorylation of GUN4 alters stimulation of MgCh activity in angiosperms (Richter et al., 2016 2016 ). The IPA1 gene encodes OsSPL14, a SBP-domain transcription factor, and promotes both yield and disease resistance in rice (Zhang H et al., 2017 2017 ; Wang J et al., 2018 2018 ). Wang B et al. ( 2018 2018 ) reported that ubiquitination and phosphorylation modifications could regulate IPA1 function at the post-transcriptional level. For example, the RING-finger E3 ligase stabilizes IPA1 in shoot apexes through K63-linked polyubiquitination, but it promotes the degradation of IPA1 in panicles through K48-linked polyubiquitination. Moreover, phosphorylated IPA1 at amino acid Ser163 activates the expression of pathogen defense gene WRKY45 , leading to enhanced disease resistance, which indicate that fine-modifying IPA1 protein leads to an “ideal” rice plant with fewer tillers, better lodging resistance, and enhanced grain yield. Thus, new directions of research are beginning to examine how we can exploit genome editing to benefit crop breeding by targeting the key sites in PTMs. Although some technical hurdles still remain to be overcome, we probably can apply CRISPR/Cas technology to simultaneously editing of multiple phosphorylation or ubiquitination sites of IPA1 or other key regulators in a precise and efficient manner, dynamic modulating of key protein activity or abundance, redirecting plant development in a multifunctional way, and providing new insights to a new level of plant synthetic biology. IPA1: ideal plant architecture 1; Glu: glutamate; Lys: lysine; Arg: arginine; Ser: serine; tRNA: transfer RNA; GluTR: Glu-tRNA reductase; GUN: GENOMES UNCOUPLED; K48/63: lysine 48/63; DEP1: dense and erect panicle 1; Ub: ubiquitination Editing for modification of protein–protein interaction 2 The term “protein–protein interaction” encompasses a variety of events, such as transient and stable complexes. The interaction between the proteins can be relevant to a variety of biological processes including metabolic and signaling pathways, stress responses, plant defense, and organismal systems (Morsy et al., 2008 2008 ; Jorrín-Novo et al., 2009 2009 ). For example, the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of glutamyl-transfer RNA (tRNA) catalyzed by glutamyl-tRNA reductase (GluTR) is the rate-limiting enzyme of 5-aminolevulinic acid (ALA) formation and chlorophyll biosynthesis (Beale, 1999 1999 ). Three proteins are known to regulate GluTR activity in chloroplasts, fluorescent (FLU) protein (Meskauskiene et al., 2001 2001 ; Goslings et al., 2004 2004 ; Kauss et al., 2012 2012 ), the GluTR-binding protein (GBP, OsGBP) (Czarnecki et al., 2011 2011 ), and the caseinolytic protease (Clp) (Apitz et al., 2016 2016 ). The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with GluTR (Zhang et al., 2015 2015 ). Three sites (Arg450, Arg485, and Arg500) in the C terminal of GluTR and three sites (Glu284, Asp291, and Asp307) in the TPR3 domain of FLU play important roles in protein–protein interaction in Arabidopsis (Zhang et al., 2015 2015 ). We can use a genome editing tool for single-base substitution to edit these sites to vary the interaction ability between GluTR and FLU in crops, which may change the ALA biosynthesis, chlorophyll biosynthesis rate, or even plant stress resistance. This paves the way for achieving functional modifications rather than total knock-out. Protoplast editing: making gene editing more applicable in plant breeding 1 In animals, genome editing is often DNA-free because guide RNA (gRNA) and Cas9 were delivered into cells in the form of pre-assembled ribonucleoproteins (RNPs) via electroporation (Kim et al., 2014 2014 ; Liang et al., 2015 2015 ). However, in plants, the presence of a cell wall makes it impossible to use transfection or electroporation for nucleic acid and/or protein delivery. Transient protoplast transfection has also been used for evaluation of gene editing reagents in various crops such as rice and tomato (Shan et al., 2014 2014 ; Čermák et al., 2015 2015 ; Woo et al., 2015 2015 ; Lin et al., 2018 2018 ), but because of the lack of a reasonably efficient protoplast regeneration system for almost all crop plants, gene editing via the protoplast seems not feasible yet for plant breeding. However, RNP-based protoplast editing has numerous advantages over the current transgenic plant-based method, and hence efforts should continue to be made to make it work in crops. Advantages of RNP-based protoplast editing 2 Transient delivery systems for transgene-free and DNA-free genome editing show a promising advantage in crop genetic modification. Based on lots of published data the mutagenesis efficiency in the protoplast looks higher than that in the plant-or tissue-based genome editing, which relies on traditional genetic engineering techniques (originally including gene guns, electroporation, and agrobacterium (Li et al., 2013 2013 ; Lin et al., 2018 2018 )). To avoid some disadvantages of the expression of Cas9 and gRNA from genome-inserted plasmid DNA, an efficient plasmid-free genome editing system has been developed using pre-assembled RNPs (containing Cas9/single-guide RNA (sgRNA)) in animal and plants (Liang et al., 2015 2015 ; Woo et al., 2015 2015 ). The new strategy achieved almost comparable efficiency to the plasmid-based transgenic systems with low off-target frequency. After transfection, the RNP can cleave the target immediately. The transcriptional or translational machinery is not required. Importantly, the RNP complex is degraded quickly, to achieve another valuable advantage with fewer off-target mutations. The rate of regenerated mutants was up to 46% in lettuce, according to the published data (Woo et al., 2015 2015 ). Sheen lab made progress with a DNA-free genome editing system with higher efficiency, approximately 99% (Sheen, pers. comm.). Multiplex gene editing by CRISPR/Cas9 provides a powerful tool for targeting members of multigene families. Although previous studies have shown that multiplex gene editing in plants is possible with CRISPR/Cas9 (Xie et al., 2015 2015 ) or CRISPR/centromere and promoter factor 1 (Cpf1) (Wang et al., 2017 2017 ), the Cas9 system requires large constructs to express multiple sgRNA cassettes, which is more laborious to construct and may cause instability and reduce transformation efficiency. Engineered CRISPR/Cpf1 with a simple short direct repeat (DR)-guide array still has the disadvantage of low cleave efficiency and off-target mutation. For pre-assembled RNPs one can synthesize multiplex sgRNAs, with, theoretically, no limitation for the RNP complex. This will demonstrate the feasibility of high-efficiency multiplex gene editing in the protoplast. Currently, most advances in CRISPR/Cas application in crops focus on single or multigene knockouts, and chromosomal deletions, which exclude some “bad” genes or Cis -elements. In wheat, mutations of three mildew-resistance locus O ( MLO ) homeologs by CRISPR/Cas9 result in improved resistance to Blumeria graminis f. sp. tritici infection (Wang et al., 2014 2014 ). Also editing of an α-Kafirin gene family increases digestibility and protein quality in sorghum (Li et al., 2018 2018 ). These strategies are based on CRISPR/Cas-targeted modification of susceptibility genes in crop species. Compared with the great progress of genome editing in animals, the big challenge in plants is in-frame gene knock-ins by the CRISPR/Cas system, which can produce a “gain of function” and facilitate breeding by introducing new alleles faster or generating allelic variants that do not exist naturally. Also, knock-in can be used to alter multiple elite traits by stacking genes in a single variety, which have great value for crop trait improvement (Chen et al., 2019 2019 ). Apparently, gene knock-in with a protoplast system shows more advantages in higher efficiency and accuracy than the traditional method. The Sheen lab at Harvard University successfully in-frame integrated the exogenous tags in Arabidopsis and the tobacco genomes (Sheen, pers. comm.), because using preassembled RNP complexes consisting of Cas9 protein and sgRNAs, the complexes were introduced directly into protoplasts using a polyethylene glycol-based method and have shown precise and efficient gene knock-out (Woo et al., 2015 2015 ) or knock-in (Sheen, pers. comm.). The previous advantages and pitfalls give us a perspective on CRISPR/Cas that might be safely used for the design of genetic modified crops as with hybrid crops. Paradigm-changing technique for protoplast regeneration 2 There is a problem in crop regeneration after gene editing in the protoplast. Though some papers claimed that they regenerated some crops from the protoplast, as is known the regeneration efficiency is far from providing a feasible application. How to regenerate a plant from a single protoplast? Actually, in animals, pluripotent stem cells (also known as induced pluripotent stem (iPS) cells) are derived directly from adult tissues, and induced by some kinases, transcription factors, or other components (Takahashi and Yamanaka, 2006 2006 ). Unlike animals, plant stem cells are innately undifferentiated cells located in the meristems of plants during the whole life cycle (Pierre-Jerome et al., 2018 2018 ). However, the tissue is limited and surrounded by differentiated cells. Importantly, these innate stem cells are changed at the cell characteristics, cell polarity, epigenetic modification, and physiological levels after removing the cell wall. Compared with animal cells, plant cells also can be induced to homogeneous embryonic stem cells in Arabidopsis and tobacco. With these cells, we can manipulate the genome rewriting easily and improve the regeneration at higher efficiency in Arabidopsis and tobacco (data not shown; rice is a potential model, which is shown in Fig. 5 5 ). Whether it works well in main crops such as rice, maize, wheat, soybean, and barley remains to be seen. It remains challenging but would be rewarding if achieved. Another question is what the protoplast-regenerated plants look like? The plants germinate from a seed and experience programed genetic and epigenetic modifications to produce environmentally adaptable plants. If we produce a somatic regenerated plant, the agronomic traits and adaptability will be doubtful, even though the reported protoplasts in regenerated tobacco look good (Lin et al., 2018 2018 ). Another group found that regeneration of Solanum tuberosum plants from protoplasts induces widespread genome instability (Fossi et al., 2019 2019 ), so more detailed information is needed before application. CRISPR/Cas is a sophisticated toolkit, but still under development and evolution. More and more researchers are working on the improvement and application of the toolkit, and such efforts should eventually lead to the implement of precision agriculture in the future. Cas: clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein; gRNA: guide RNA; RNP: ribonucleoprotein References MA Al-Zeer , M Dutkiewicz , A von Hacht , 等 . Alternatively spliced variants of the 5'-UTR of the ARPC2 mRNA regulate translation by an internal ribosome entry site (IRES) harboring a guanine-quadruplex motif . RNA Biol , 2019 . 16 ( 11 ): 1622 - 1632 . 31387452 DOI: 10.1080/15476286.2019.1652524 http://doi.org/10.1080/15476286.2019.1652524 . Alternatively spliced variants of the 5'-UTR of the ARPC2 mRNA regulate translation by an internal ribosome entry site (IRES) harboring a guanine-quadruplex motif J Apitz , K Nishimura , J Schmied , 等 . Posttranslational control of ALA synthesis includes GluTR degradation by Clp protease and stabilization by GluTR-binding protein . Plant Physiol , 2016 . 170 ( 4 ): 2040 - 2051 . 26884485 DOI: 10.1104/pp.15.01945 http://doi.org/10.1104/pp.15.01945 . Posttranslational control of ALA synthesis includes GluTR degradation by Clp protease and stabilization by GluTR-binding protein SI Beale . Enzymes of chlorophyll biosynthesis . Photosynth Res , 1999 . 60 ( 1 ): 43 - 73 . DOI: 10.1023/a:1006297731456 http://doi.org/10.1023/a:1006297731456 . Enzymes of chlorophyll biosynthesis E Butelli , C Licciardello , Y Zhang , 等 . Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges . Plant Cell , 2012 . 24 ( 3 ): 1242 - 1255 . 22427337 DOI: 10.1105/tpc.111.095232 http://doi.org/10.1105/tpc.111.095232 . Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges T Cermak , EL Doyle , M Christian , 等 . Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting . Nucleic Acids Res , 2011 . 39 ( 12 ): e82 21493687 DOI: 10.1093/nar/gkr218 http://doi.org/10.1093/nar/gkr218 . Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting T Čermák , NJ Baltes , R Čegan , 等 . High-frequency, precise modification of the tomato genome . Genome Biol, 16:232 , 2015 . DOI: 10.1186/s13059-015-0796-9 http://doi.org/10.1186/s13059-015-0796-9 . High-frequency, precise modification of the tomato genome A Chavez , J Scheiman , S Vora , 等 . Highly efficient Cas9-mediated transcriptional programming . Nat Methods , 2015 . 12 ( 4 ): 326 - 328 . 25730490 DOI: 10.1038/nmeth.3312 http://doi.org/10.1038/nmeth.3312 . Highly efficient Cas9-mediated transcriptional programming KL Chen , YP Wang , R Zhang , 等 . CRISPR/Cas genome editing and precision plant breeding in agriculture . Annu Rev Plant Biol , 2019 . 70 667 - 697 . 30835493 DOI: 10.1146/annurev-arplant-050718-100049 http://doi.org/10.1146/annurev-arplant-050718-100049 . CRISPR/Cas genome editing and precision plant breeding in agriculture O Czarnecki , B Hedtke , M Melzer , 等 . An Arabidopsis GluTR binding protein mediates spatial separation of 5-aminolevulinic acid synthesis in chloroplasts . Plant Cell , 2011 . 23 ( 12 ): 4476 - 4491 . 22180625 DOI: 10.1105/tpc.111.086421 http://doi.org/10.1105/tpc.111.086421 . An Arabidopsis GluTR binding protein mediates spatial separation of 5-aminolevulinic acid synthesis in chloroplasts X Deng , XF Cao . Roles of pre-mRNA splicing and polyadenylation in plant development . Curr Opin Plant Biol , 2017 . 35 45 - 53 . 27866125 DOI: 10.1016/j.pbi.2016.11.003 http://doi.org/10.1016/j.pbi.2016.11.003 . Roles of pre-mRNA splicing and polyadenylation in plant development YL Deribe , T Pawson , I Dikic . Post-translational modifications in signal integration . Nat Struct Mol Biol , 2010 . 17 ( 6 ): 666 - 672 . 20495563 DOI: 10.1038/nsmb.1842 http://doi.org/10.1038/nsmb.1842 . Post-translational modifications in signal integration GY Duan , D Walther . The roles of post-translational modifications in the context of protein interaction networks . PLoS Comput Biol , 2015 . 11 ( 2 ): e1004049 25692714 DOI: 10.1371/journal.pcbi.1004049 http://doi.org/10.1371/journal.pcbi.1004049 . The roles of post-translational modifications in the context of protein interaction networks A Endo , M Masafumi , H Kaya , 等 . Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida . Sci Rep, 6:38169 , 2016 . DOI: 10.1038/srep38169 http://doi.org/10.1038/srep38169 . Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida I Eş , M Gavahian , FJ Marti-Quijal , 等 . The application of the CRISPR-Cas9 genome editing machinery in food and agricultural science: current status, future perspectives, and associated challenges . Biotechnol Adv , 2019 . 37 ( 3 ): 410 - 421 . 30779952 DOI: 10.1016/j.biotechadv.2019.02.006 http://doi.org/10.1016/j.biotechadv.2019.02.006 . The application of the CRISPR-Cas9 genome editing machinery in food and agricultural science: current status, future perspectives, and associated challenges RV Espley , C Brendolise , D Chagné , 等 . Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples . Plant Cell , 2009 . 21 ( 1 ): 168 - 183 . 19151225 DOI: 10.1105/tpc.108.059329 http://doi.org/10.1105/tpc.108.059329 . Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples S Filichkin , HD Priest , M Megraw , 等 . Alternative splicing in plants: directing traffic at the crossroads of adaptation and environmental stress . Curr Opin Plant Biol , 2015 . 24 125 - 135 . 25835141 DOI: 10.1016/j.pbi.2015.02.008 http://doi.org/10.1016/j.pbi.2015.02.008 . Alternative splicing in plants: directing traffic at the crossroads of adaptation and environmental stress M Fossi , K Amundson , S Kuppu , 等 . Regeneration of Solanum tuberosum plants from protoplasts induces widespread genome instability . Plant Physiol , 2019 . 180 78 - 86 . 30792232 DOI: 10.1104/pp.18.00906 http://doi.org/10.1104/pp.18.00906 . Regeneration of Solanum tuberosum plants from protoplasts induces widespread genome instability D Goslings , R Meskauskiene , C Kim , 等 . Concurrent interactions of heme and FLU with Glu tRNA reductase (HEMA1), the target of metabolic feedback inhibition of tetrapyrrole biosynthesis, in dark- and light-grown Arabidopsis plants . Plant J , 2004 . 40 ( 6 ): 957 - 967 . 15584960 DOI: 10.1111/j.1365-313x.2004.02262.x http://doi.org/10.1111/j.1365-313x.2004.02262.x . Concurrent interactions of heme and FLU with Glu tRNA reductase (HEMA1), the target of metabolic feedback inhibition of tetrapyrrole biosynthesis, in dark- and light-grown Arabidopsis plants LT Hickey , AN Hafeez , H Robinson , 等 . Breeding crops to feed 10 billion . Nat Biotechnol , 2019 . 37 ( 2 ): 744 - 754 . 31209375 DOI: 10.1038/s41587-019-0152-9 http://doi.org/10.1038/s41587-019-0152-9 . Breeding crops to feed 10 billion JH Hu , SM Miller , MH Geurts , 等 . Evolved Cas9 variants with broad PAM compatibility and high DNA specificity . Nature , 2018 . 556 ( 7699 ): 57 - 63 . 29512652 DOI: 10.1038/nature26155 http://doi.org/10.1038/nature26155 . Evolved Cas9 variants with broad PAM compatibility and high DNA specificity K Hua , XP Tao , JK Zhu . Expanding the base editing scope in rice by using Cas9 variants . Plant Biotechnol J , 2018 . 17 ( 2 ): 499 - 504 . 30051586 DOI: 10.1111/pbi.12993 http://doi.org/10.1111/pbi.12993 . Expanding the base editing scope in rice by using Cas9 variants K Hua , JS Zhang , JR Botella , 等 . Perspectives on the application of genome-editing technologies in crop breeding . Mol Plant , 2019 . 12 ( 8 ): 1047 - 1059 . 31260812 DOI: 10.1016/j.molp.2019.06.009 http://doi.org/10.1016/j.molp.2019.06.009 . Perspectives on the application of genome-editing technologies in crop breeding SC Huber , SC Hardin . Numerous posttranslational modifications provide opportunities for the intricate regulation of metabolic enzymes at multiple levels . Curr Opin Plant Biol , 2004 . 7 ( 3 ): 318 - 322 . 15134753 DOI: 10.1016/j.pbi.2004.03.002 http://doi.org/10.1016/j.pbi.2004.03.002 . Numerous posttranslational modifications provide opportunities for the intricate regulation of metabolic enzymes at multiple levels AG Hunt . The Arabidopsis polyadenylation factor subunit CPSF30 as conceptual link between mRNA polyadenylation and cellular signaling . Curr Opin Plant Biol , 2014 . 21 128 - 132 . 25104048 DOI: 10.1016/j.pbi.2014.07.002 http://doi.org/10.1016/j.pbi.2014.07.002 . The Arabidopsis polyadenylation factor subunit CPSF30 as conceptual link between mRNA polyadenylation and cellular signaling HG Jia , YZ Zhang , V Orbović , 等 . Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker . Plant Biotechnol J , 2017 . 15 ( 7 ): 817 - 823 . 27936512 DOI: 10.1111/pbi.12677 http://doi.org/10.1111/pbi.12677 . Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker M Jiang , YH Liu , RQ Li , 等 . A suppressor mutation partially reverts the xantha trait via lowered methylation in the promoter of genomes uncoupled 4 in Rice . Front Plant Sci, 10:1003 , 2019 . DOI: 10.3389/fpls.2019.01003 http://doi.org/10.3389/fpls.2019.01003 . A suppressor mutation partially reverts the xantha trait via lowered methylation in the promoter of genomes uncoupled 4 in Rice YQ Jiao , YH Wang , DW Xue , 等 . Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice . Nat Genet , 2010 . 42 ( 6 ): 541 - 544 . 20495565 DOI: 10.1038/ng.591 http://doi.org/10.1038/ng.591 . Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice JV Jorrín-Novo , AM Maldonado , S Echevarria-Zomeno , 等 . Plant proteomics update (2007–2008): second-generation proteomic techniques, an appropriate experimental design, and data analysis to fulfill MIAPE standards, increase plant proteome coverage and expand biological knowledge . J Proteomics , 2009 . 72 ( 3 ): 285 - 314 . 19367730 DOI: 10.1016/j.jprot.2009.01.026 http://doi.org/10.1016/j.jprot.2009.01.026 . Plant proteomics update (2007–2008): second-generation proteomic techniques, an appropriate experimental design, and data analysis to fulfill MIAPE standards, increase plant proteome coverage and expand biological knowledge AP Kausch , K Nelson-Vasilchik , J Hague , 等 . Edit at will: genotype independent plant transformation in the era of advanced genomics and genome editing . Plant Sci , 2019 . 281 186 - 205 . 30824051 DOI: 10.1016/j.plantsci.2019.01.006 http://doi.org/10.1016/j.plantsci.2019.01.006 . Edit at will: genotype independent plant transformation in the era of advanced genomics and genome editing D Kauss , S Bischof , S Steiner , 等 . FLU, a negative feedback regulator of tetrapyrrole biosynthesis, is physically linked to the final steps of the Mg ++ -branch of this pathway . FEBS Lett , 2012 . 586 ( 3 ): 211 - 216 . 22212719 DOI: 10.1016/j.febslet.2011.12.029 http://doi.org/10.1016/j.febslet.2011.12.029 . FLU, a negative feedback regulator of tetrapyrrole biosynthesis, is physically linked to the final steps of the Mg++-branch of this pathway S Kim , D Kim , SW Cho , 等 . Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins . Genome Res , 2014 . 24 ( 6 ): 1012 - 1019 . 24696461 DOI: 10.1101/gr.171322.113 http://doi.org/10.1101/gr.171322.113 . Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins BP Kleinstiver , SQ Tsai , MS Prew , 等 . Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells . Nat Biotechnol , 2016 . 34 ( 8 ): 869 - 874 . 27347757 DOI: 10.1038/nbt.3620 http://doi.org/10.1038/nbt.3620 . Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells K Leppek , R Das , M Barna . Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them . Nat Rev Mol Cell , 2018 . 19 ( 3 ): 158 - 174 . DOI: 10.1038/nrm.2017.103 http://doi.org/10.1038/nrm.2017.103 . Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them AX Li , SG Jia , A Yobi , 等 . Editing of an alpha-kafirin gene family increases, digestibility and protein quality in sorghum . Plant Physiol , 2018 . 177 ( 4 ): 1425 - 1438 . 29925584 DOI: 10.1104/pp.18.00200 http://doi.org/10.1104/pp.18.00200 . Editing of an alpha-kafirin gene family increases, digestibility and protein quality in sorghum JF Li , JE Norville , J Aach , 等 . Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 . Nat Biotechnol , 2013 . 31 ( 8 ): 688 - 691 . 23929339 DOI: 10.1038/nbt.2654 http://doi.org/10.1038/nbt.2654 . Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 T Li , B Liu , MH Spalding , 等 . High-efficiency TALEN-based gene editing produces disease-resistant rice . Nat Biotechnol , 2012 . 30 ( 5 ): 390 - 392 . 22565958 DOI: 10.1038/nbt.2199 http://doi.org/10.1038/nbt.2199 . High-efficiency TALEN-based gene editing produces disease-resistant rice T Li , B Liu , CY Chen , 等 . TALEN-mediated homologous recombination produces site-directed DNA base change and herbicide-resistant rice . J Genet Genomic , 2016 . 43 ( 5 ): 297 - 305 . DOI: 10.1016/j.jgg.2016.03.005 http://doi.org/10.1016/j.jgg.2016.03.005 . TALEN-mediated homologous recombination produces site-directed DNA base change and herbicide-resistant rice WT Li , ZW Zhu , M Chern , 等 . A natural allele of a transcription factor in rice confers broad-spectrum blast resistance . Cell , 2017 . 170 ( 1 ): 114 - 126 . 28666113 DOI: 10.1016/j.cell.2017.06.008 http://doi.org/10.1016/j.cell.2017.06.008 . A natural allele of a transcription factor in rice confers broad-spectrum blast resistance YB Li , CC Fan , YZ Xing , 等 . Natural variation in GS5 plays an important role in regulating grain size and yield in rice . Nat Genet , 2011 . 43 ( 12 ): 1266 - 1269 . 22019783 DOI: 10.1038/ng.977 http://doi.org/10.1038/ng.977 . Natural variation in GS5 plays an important role in regulating grain size and yield in rice XQ Liang , J Potter , S Kumar , 等 . Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection . J Biotechnol , 2015 . 208 44 - 53 . 26003884 DOI: 10.1016/j.jbiotec.2015.04.024 http://doi.org/10.1016/j.jbiotec.2015.04.024 . Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection CS Lin , CT Hsu , LH Yang , 等 . Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration . Plant Biotechnol J , 2018 . 16 ( 7 ): 1295 - 1310 . 29230929 DOI: 10.1111/pbi.12870 http://doi.org/10.1111/pbi.12870 . Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration S Lin , YY Zhao , YF Zhu , 等 . An effective and inducible system of TAL effector-mediated transcriptional repression in Arabidopsis . Mol Plant , 2016 . 9 ( 11 ): 1546 - 1549 . 27677459 DOI: 10.1016/j.molp.2016.09.003 http://doi.org/10.1016/j.molp.2016.09.003 . An effective and inducible system of TAL effector-mediated transcriptional repression in Arabidopsis SM Liu , J Jiang , Y Liu , 等 . Characterization and evaluation of OsLCT1 and OsNramp5 mutants generated through CRISPR/Cas9-mediated mutagenesis for breeding low Cd rice . Rice Sci , 2019 . 26 ( 2 ): 88 - 97 . DOI: 10.1016/j.rsci.2019.01.002 http://doi.org/10.1016/j.rsci.2019.01.002 . Characterization and evaluation of OsLCT1 and OsNramp5 mutants generated through CRISPR/Cas9-mediated mutagenesis for breeding low Cd rice A Lloyd , CL Plaisier , D Carroll , 等 . Targeted mutagenesis using zinc-finger nucleases in Arabidopsis . Proc Natl Acad Sci USA , 2005 . 102 ( 6 ): 2232 - 2237 . DOI: 10.1073/pnas.0409339102 http://doi.org/10.1073/pnas.0409339102 . Targeted mutagenesis using zinc-finger nucleases in Arabidopsis MM Mahfouz , LX Li , M Shamimuzzaman , 等 . De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks . Proc Natl Acad Sci USA , 2011 . 108 ( 6 ): 2623 - 2628 . DOI: 10.1073/pnas.1019533108 http://doi.org/10.1073/pnas.1019533108 . De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks YF Mao , JR Botella , YG Liu , 等 . Gene editing in plants: progress and challenges . Natl Sci Rev , 2019 . 6 ( 3 ): 421 - 437 . DOI: 10.1093/nsr/nwz005 http://doi.org/10.1093/nsr/nwz005 . Gene editing in plants: progress and challenges R Meskauskiene , M Nater , D Goslings , 等 . FLU: a negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana . Proc Natl Acad Sci USA , 2001 . 98 ( 22 ): 12826 - 12831 . DOI: 10.1073/pnas.221252798 http://doi.org/10.1073/pnas.221252798 . FLU: a negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana B Minkenberg , KB Xie , YN Yang . Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes . Plant J , 2017 . 89 ( 3 ): 636 - 648 . 27747971 DOI: 10.1111/tpj.13399 http://doi.org/10.1111/tpj.13399 . Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes KA Molla , YN Yang . CRISPR/Cas-mediated base editing: technical considerations and practical applications . Trends Biotechnol , 2019 . 37 ( 10 ): 1121 - 1142 . 30995964 DOI: 10.1016/j.tibtech.2019.03.008 http://doi.org/10.1016/j.tibtech.2019.03.008 . CRISPR/Cas-mediated base editing: technical considerations and practical applications M Morsy , S Gouthu , S Orchard , 等 . Charting plant interactomes: possibilities and challenges . Trends Plant Sci , 2008 . 13 ( 4 ): 183 - 191 . 18329319 DOI: 10.1016/j.tplants.2008.01.006 http://doi.org/10.1016/j.tplants.2008.01.006 . Charting plant interactomes: possibilities and challenges V Nekrasov , B Staskawicz , D Weigel , 等 . Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease . Nat Biotechnol , 2013 . 31 ( 8 ): 691 - 693 . 23929340 DOI: 10.1038/nbt.2655 http://doi.org/10.1038/nbt.2655 . Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease H Nishimasu , X Shi , S Ishiguro , 等 . Engineered CRISPR-Cas9 nuclease with expanded targeting space . Science , 2018 . 361 ( 6408 ): 1259 - 1262 . 30166441 DOI: 10.1126/science.aas9129 http://doi.org/10.1126/science.aas9129 . Engineered CRISPR-Cas9 nuclease with expanded targeting space T Oikawa , H Maeda , T Oguchi , 等 . The birth of a black rice gene and its local spread by introgression . Plant Cell , 2015 . 27 ( 9 ): 2401 - 2414 . 26362607 DOI: 10.1105/tpc.15.00310 http://doi.org/10.1105/tpc.15.00310 . The birth of a black rice gene and its local spread by introgression R Pandiarajan , A Grover . In vivo promoter engineering in plants: are we ready? . Plant Sci , 2018 . 277 132 - 138 . 30466578 DOI: 10.1016/j.plantsci.2018.10.011 http://doi.org/10.1016/j.plantsci.2018.10.011 . In vivo promoter engineering in plants: are we ready? A Piatek , Z Ali , H Baazim , 等 . RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors . Plant Biotechnol J , 2015 . 13 ( 4 ): 578 - 589 . 25400128 DOI: 10.1111/pbi.12284 http://doi.org/10.1111/pbi.12284 . RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors E Pierre-Jerome , C Drapek , PN Benfey . Regulation of division and differentiation of plant stem cells . Annu Rev Cell Dev Biol , 2018 . 34 ( 1 ): 289 - 310 . 30134119 DOI: 10.1146/annurev-cellbio-100617-062459 http://doi.org/10.1146/annurev-cellbio-100617-062459 . Regulation of division and differentiation of plant stem cells ZR Qin , JJ Wu , SF Geng , 等 . Regulation of FT splicing by an endogenous cue in temperate grasses . Nat Commun, 8:14320 , 2017 . DOI: 10.1038/ncomms14320 http://doi.org/10.1038/ncomms14320 . Regulation of FT splicing by an endogenous cue in temperate grasses ASN Reddy , Y Marquez , M Kalyna , 等 . Complexity of the alternative splicing landscape in plants . Plant Cell , 2013 . 25 ( 10 ): 3657 - 3683 . 24179125 DOI: 10.1105/tpc.113.117523 http://doi.org/10.1105/tpc.113.117523 . Complexity of the alternative splicing landscape in plants B Ren , L Liu , SF Li , 等 . Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice . Mol Plant , 2019 . 12 ( 7 ): 1015 - 1026 . 30928635 DOI: 10.1016/j.molp.2019.03.010 http://doi.org/10.1016/j.molp.2019.03.010 . Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice AS Richter , C Hochheuser , C Fufezan , 等 . Phosphorylation of GENOMES UNCOUPLED 4 alters stimulation of Mg chelatase activity in angiosperms . Plant Physiol , 2016 . 172 ( 3 ): 1578 - 1595 . 27688621 DOI: 10.1104/pp.16.01036 http://doi.org/10.1104/pp.16.01036 . Phosphorylation of GENOMES UNCOUPLED 4 alters stimulation of Mg chelatase activity in angiosperms D Rodriguez-Leal , ZH Lemmon , J Man , 等 . Engineering quantitative trait variation for crop improvement by genome editing . Cell , 2017 . 171 ( 2 ): 470 - 480E8 . 28919077 DOI: 10.1016/j.cell.2017.08.030 http://doi.org/10.1016/j.cell.2017.08.030 . Engineering quantitative trait variation for crop improvement by genome editing QW Shan , YP Wang , J Li , 等 . Targeted genome modification of crop plants using a CRISPR-Cas system . Nat Biotechnol , 2013 . 31 ( 8 ): 686 - 688 . 23929338 DOI: 10.1038/nbt.2650 http://doi.org/10.1038/nbt.2650 . Targeted genome modification of crop plants using a CRISPR-Cas system QW Shan , YP Wang , J Li , 等 . Genome editing in rice and wheat using the CRISPR/Cas system . Nat Protoc , 2014 . 9 ( 10 ): 2395 - 2410 . 25232936 DOI: 10.1038/nprot.2014.157 http://doi.org/10.1038/nprot.2014.157 . Genome editing in rice and wheat using the CRISPR/Cas system QW Shan , Y Zhang , KL Chen , 等 . Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology . Plant Biotechnol J , 2015 . 13 ( 6 ): 791 - 800 . 25599829 DOI: 10.1111/pbi.12312 http://doi.org/10.1111/pbi.12312 . Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology JR Shi , HR Gao , HY Wang , 等 . ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions . Plant Biotechnol J , 2017 . 15 ( 2 ): 207 - 216 . 27442592 DOI: 10.1111/pbi.12603 http://doi.org/10.1111/pbi.12603 . ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions Z Shimatani , S Kashojiya , M Takayama , 等 . Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion . Nat Biotechnol , 2017 . 35 ( 5 ): 441 - 443 . 28346401 DOI: 10.1038/nbt.3833 http://doi.org/10.1038/nbt.3833 . Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion S Soyk , NA Müller , SJ Park , 等 . Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato . Nat Genet , 2017 . 49 ( 1 ): 162 - 168 . 27918538 DOI: 10.1038/ng.3733 http://doi.org/10.1038/ng.3733 . Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato YW Sun , GA Jiao , ZP Liu , 等 . Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes . Front Plant Sci, 8:298 , 2017 . DOI: 10.3389/fpls.2017.00298 http://doi.org/10.3389/fpls.2017.00298 . Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes K Takahashi , S Yamanaka . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors . Cell , 2006 . 126 ( 4 ): 663 - 676 . 16904174 DOI: 10.1016/j.cell.2006.07.024 http://doi.org/10.1016/j.cell.2006.07.024 . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors M Takenaka , A Zehrmann , D Verbitskiy , 等 . RNA editing in plants and its evolution . Annu Rev Genet , 2013 . 47 335 - 352 . 24274753 DOI: 10.1146/annurev-genet-111212-133519 http://doi.org/10.1146/annurev-genet-111212-133519 . RNA editing in plants and its evolution L Tang , BG Mao , YK Li , 等 . Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield . Sci Rep, 7:14438 , 2017 . DOI: 10.1038/s41598-017-14832-9 http://doi.org/10.1038/s41598-017-14832-9 . Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield X Tang , LG Lowder , T Zhang , 等 . A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants . Nat Plants , 2017 . 3 ( 3 ): 17018 28211909 DOI: 10.1038/nplants.2017.18 http://doi.org/10.1038/nplants.2017.18 . A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants AG von Arnim , QD Jia , JN Vaughn . Regulation of plant translation by upstream open reading frames . Plant Sci , 2014 . 214 1 - 12 . 24268158 DOI: 10.1016/j.plantsci.2013.09.006 http://doi.org/10.1016/j.plantsci.2013.09.006 . Regulation of plant translation by upstream open reading frames B Wang , SM Smith , JY Li . Genetic regulation of shoot architecture . Annu Rev Plant Biol , 2018 . 69 ( 1 ): 437 - 468 . 29553800 DOI: 10.1146/annurev-arplant-042817-040422 http://doi.org/10.1146/annurev-arplant-042817-040422 . Genetic regulation of shoot architecture FJ Wang , CL Wang , PQ Liu , 等 . Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922 . PLoS ONE , 2016 . 11 ( 4 ): e0154027 27116122 DOI: 10.1371/journal.pone.0154027 http://doi.org/10.1371/journal.pone.0154027 . Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922 J Wang , L Zhou , H Shi , 等 . A single transcription factor promotes both yield and immunity in rice . Science , 2018 . 361 ( 6406 ): 1026 - 1028 . 30190406 DOI: 10.1126/science.aat7675 http://doi.org/10.1126/science.aat7675 . A single transcription factor promotes both yield and immunity in rice MG Wang , YF Mao , YM Lu , 等 . Multiplex gene editing in rice using the CRISPR-Cpf1 system . Mol Plant , 2017 . 10 ( 7 ): 1011 - 1013 . 28315752 DOI: 10.1016/j.molp.2017.03.001 http://doi.org/10.1016/j.molp.2017.03.001 . Multiplex gene editing in rice using the CRISPR-Cpf1 system SK Wang , K Wu , QB Yuan , 等 . Control of grain size, shape and quality by OsSPL16 in rice . Nat Genet , 2012 . 44 ( 8 ): 950 - 954 . 22729225 DOI: 10.1038/ng.2327 http://doi.org/10.1038/ng.2327 . Control of grain size, shape and quality by OsSPL16 in rice SK Wang , S Li , Q Liu , 等 . The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality . Nat Genet , 2015 . 47 ( 8 ): 949 - 954 . 26147620 DOI: 10.1038/ng.3352 http://doi.org/10.1038/ng.3352 . The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality YP Wang , X Cheng , QW Shan , 等 . Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew . Nat Biotechnol , 2014 . 32 ( 9 ): 947 - 951 . 25038773 DOI: 10.1038/nbt.2969 http://doi.org/10.1038/nbt.2969 . Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew JW Woo , J Kim , SI Kwon , 等 . DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins . Nat Biotechnol , 2015 . 33 ( 11 ): 1162 - 1164 . 26479191 DOI: 10.1038/nbt.3389 http://doi.org/10.1038/nbt.3389 . DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins L Wu , HY Zhou , QQ Zhang , 等 . DNA methylation mediated by a microRNA pathway . Mol Cell , 2010 . 38 ( 3 ): 465 - 475 . 20381393 DOI: 10.1016/j.molcel.2010.03.008 http://doi.org/10.1016/j.molcel.2010.03.008 . DNA methylation mediated by a microRNA pathway KB Xie , B Minkenberg , YN Yang . Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system . Proc Natl Acad Sci USA , 2015 . 112 ( 11 ): 3570 - 3575 . DOI: 10.1073/pnas.1420294112 http://doi.org/10.1073/pnas.1420294112 . Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system CJ Xu , Y Liu , YB Li , 等 . Differential expression of GS5 regulates grain size in rice . J Exp Bot , 2015 . 66 ( 9 ): 2611 - 2623 . 25711711 DOI: 10.1093/jxb/erv058 http://doi.org/10.1093/jxb/erv058 . Differential expression of GS5 regulates grain size in rice RF Xu , YC Yang , RY Qing , 等 . Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice . J Genet Genomics , 2016 . 43 ( 8 ): 529 - 532 . 27543262 DOI: 10.1016/j.jgg.2016.07.003 http://doi.org/10.1016/j.jgg.2016.07.003 . Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice CX Xue , HW Zhang , QP Lin , 等 . Manipulating mRNA splicing by base editing in plants . Sci China Life Sci , 2018 . 61 ( 11 ): 1293 - 1300 . 30267262 DOI: 10.1007/s11427-018-9392-7 http://doi.org/10.1007/s11427-018-9392-7 . Manipulating mRNA splicing by base editing in plants RX Yang , PC Li , HL Mei , 等 . Fine-tuning of miR528 accumulation modulates flowering time in rice . Mol Plant , 2019 . 12 ( 8 ): 1103 - 1113 . 31059825 DOI: 10.1016/j.molp.2019.04.009 http://doi.org/10.1016/j.molp.2019.04.009 . Fine-tuning of miR528 accumulation modulates flowering time in rice AJ Ytterberg , ON Jensen . Modification-specific proteomics in plant biology . J Proteomics , 2010 . 73 ( 11 ): 2249 - 2266 . 20541636 DOI: 10.1016/j.jprot.2010.06.002 http://doi.org/10.1016/j.jprot.2010.06.002 . Modification-specific proteomics in plant biology SSA Zaidi , MS Mukhtar , S Mansoor . Genome editing: targeting susceptibility genes for plant disease resistance . Trends Biotechnol , 2018 . 36 ( 9 ): 898 - 906 . 29752192 DOI: 10.1016/j.tibtech.2018.04.005 http://doi.org/10.1016/j.tibtech.2018.04.005 . Genome editing: targeting susceptibility genes for plant disease resistance B Zetsche , JS Gootenberg , OO Abudayyeh , 等 . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system . Cell , 2015 . 163 ( 3 ): 759 - 771 . 26422227 DOI: 10.1016/j.cell.2015.09.038 http://doi.org/10.1016/j.cell.2015.09.038 . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system H Zhang , JS Zhang , ZB Lang , 等 . Genome editing-principles and applications for functional genomics research and crop improvement . Plant Sci , 2017 . 36 ( 4 ): 291 - 309 . DOI: 10.1080/07352689.2017.1402989 http://doi.org/10.1080/07352689.2017.1402989 . Genome editing-principles and applications for functional genomics research and crop improvement HW Zhang , XM Si , X Ji , 等 . Genome editing of upstream open reading frames enables translational control in plants . Nat Biotechnol , 2018 . 36 ( 9 ): 894 - 898 . 30080209 DOI: 10.1038/nbt.4202 http://doi.org/10.1038/nbt.4202 . Genome editing of upstream open reading frames enables translational control in plants JS Zhang , H Zhang , JR Botella , 等 . Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties . J Integr Plant Biol , 2018 . 60 ( 5 ): 369 - 375 . 29210506 DOI: 10.1111/jipb.12620 http://doi.org/10.1111/jipb.12620 . Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties L Zhang , H Yu , B Ma , 等 . A natural tandem array alleviates epigenetic repression of IPA1 and leads to superior yielding rice . Nat Commun, 8:14789 , 2017 . DOI: 10.1038/ncomms14789 http://doi.org/10.1038/ncomms14789 . A natural tandem array alleviates epigenetic repression of IPA1 and leads to superior yielding rice M Zhang , FL Zhang , Y Fang , 等 . The non-canonical tetratricopeptide repeat (TPR) domain of fluorescent (FLU) mediates complex formation with glutamyl-tRNA reductase . J Biol Chem , 2015 . 290 ( 28 ): 17559 - 17565 . 26037924 DOI: 10.1074/jbc.M115.662981 http://doi.org/10.1074/jbc.M115.662981 . The non-canonical tetratricopeptide repeat (TPR) domain of fluorescent (FLU) mediates complex formation with glutamyl-tRNA reductase YX Zhang , AA Malzahn , S Sretenovic , 等 . The emerging and uncultivated potential of CRSIPR technology in plant science . Nat Plants , 2019 . 5 ( 8 ): 778 - 794 . 31308503 DOI: 10.1038/s41477-019-0461-5 http://doi.org/10.1038/s41477-019-0461-5 . The emerging and uncultivated potential of CRSIPR technology in plant science JP Zhou , KJ Deng , Y Cheng , 等 . CRISPR-Cas9 based genome editing reveals new insights into microRNA function and regulation in rice . Front Plant Sci, 8:1598 , 2017 . DOI: 10.3389/fpls.2017.01598 http://doi.org/10.3389/fpls.2017.01598 . CRISPR-Cas9 based genome editing reveals new insights into microRNA function and regulation in rice X Zhou , L Deng , Q Wang , 等 . Breeding of waxy rice by genome editing . Mol Plant Breed , 2018 . 16 ( 17 ): 5608 - 5615 . (in Chinese) . Breeding of waxy rice by genome editing T Zimny , S Sowa , A Tyczewska , 等 . Certain new plant breeding techniques and their marketability in the context of EU GMO legislation–recent developments . New Biotechnol , 2019 . 51 49 - 56 . DOI: 10.1016/j.nbt.2019.02.003 http://doi.org/10.1016/j.nbt.2019.02.003 . Certain new plant breeding techniques and their marketability in the context of EU GMO legislation–recent developments Y Zong , QN Song , C Li , 等 . Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A . Nat Biotechnol , 2018 . 36 ( 10 ): 950 - 953 . DOI: 10.1038/nbt.4261 http://doi.org/10.1038/nbt.4261 . Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A We thank Dr. Jen SHEEN (Department of Molecular Biology in Massachusetts General Hospital, Harvard University, USA) for sharing her working progress on the protoplast editing in lettuce, Arabidopsis, and tobacco. Compliance with ethics guidelines Yuan-yuan TAN, Hao DU, Xia WU, Yan-hua LIU, Meng JIANG, Shi-yong SONG, Liang WU, and Qing-yao SHU declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of authors. 10.1631/jzus.B1900633 Project supported by the Zhejiang Provincial S&T Project on Breeding Agricultural (Food) Crops (No. 2016C02050-2) and the National Natural Science Foundation of China (No. 31701394) 2019-11-06 2019-12-18 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 21