Search

Functional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time-consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence-guided nicking endonuclease (UNiE)-mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15-nt 3' single-strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination-mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII-UNiE. Using TGSII-UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII-UNiE. In conclusion, UNiEDA is an efficient, convenient and low-cost method for DNA cloning and multigene stacking, and the TGSII-UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.

Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

Due to its fast deterioration, soybean (Glycine max L.) has an inherently poor seed vigor. Vigor loss occurring during storage is one of the main obstacles to soybean production in the tropics. To analyze the genetic background of seed vigor, soybean seeds of a recombinant inbred line (RIL) population derived from the cross between Zhonghuang24 (ZH24, low vigor cultivar) and Huaxia3hao (HX3, vigorous cultivar) were utilized to identify the quantitative trait loci (QTLs) underlying the seed vigor under −20 °C conservation and accelerated aging conditions. According to the linkage analysis, multiple seed vigor-related QTLs were identified under both −20 °C and accelerated aging storage. Two major QTLs and eight QTL hotspots localized on chromosomes 3, 6, 9, 11, 15, 16, 17, and 19 were detected that were associated with seed vigor across two storage conditions. The indicators of seed vigor did not correlate well between the two aging treatments, and no common QTLs were detected in RIL populations stored in two conditions. These results indicated that deterioration under accelerated aging conditions was not reflective of natural aging at −20 °C. Additionally, we suggest 15 promising candidate genes that could possibly determine the seed vigor in soybeans, which would help explore the mechanisms responsible for maintaining high seed vigor.

The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for further improvement of CRISPR/Cas systems. The original Cas9 protein from Streptococcus canis (ScCas9) can recognize simple NNG-protospacer adjacent motif (PAM) targets, and therefore possesses a broader range relative to current CRISPR/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9+ and ScCas9++ variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9+ and ScCas9++ and a nickase variant ScCas9n++ to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice (Oryza sativa L.). This analysis revealed that ScCas9++ has higher editing efficiency than ScCas9 and ScCas9+ in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n++ to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n++ . This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C- 1 -C17 ) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (three targets) and OsEui1 (two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9++ represents a crucial new tool for improving plant editing.

CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5′-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.

Golden2 (G2), a member of the GARP transcription factor superfamily, regulates several biological processes and phytohormone signaling pathways in plants. In this study, we used a rice codon-optimized maize G2 gene (rZmG2) to improve the regeneration efficiency of rice and maize calli for genetic transformation. We isolated a promoter driving strong and callus-specific expression from rice to drive rZmG2 transcription from a transgene after transformation of two indica and two japonica rice cultivars. The resulting rZmG2 transgenic calli turned green in advance at the differentiation stage, thus significantly raising the regeneration rates of the transgenic indica and japonica rice plants relative to control transformations. Similar effect of this gene on improving maize transformation was also observed. Transcriptome sequencing and RT-qPCR analyses showed that many rice genes related to chloroplast development and phytohormones are upregulated in rZmG2-transgenic calli. These results demonstrate that rZmG2 can promote embryogenic callus differentiation and improve regeneration efficiency by activating chloroplast development and phytohormone pathways. We also established a heat-inducible Cre/loxP-based gene-excision system to remove rZmG2 and the antibiotic selectable gene after obtaining the transgenic plants. This study provides a useful tool for functional genomics work and biotechnology in plants.

Summary Rice (Oryza sativa) is a short‐day (SD) plant originally having strong photoperiod sensitivity (PS), with SDs promoting and long days (LDs) suppressing flowering. Although the evolution of PS in rice has been extensively studied, there are few studies that combine the genetic effects and underlying mechanism of different PS gene combinations with variations in PS.We created a set of isogenic lines among the core PS‐flowering genes Hd1, Ghd7 and DTH8 using CRISPR mutagenesis, to systematically dissect their genetic relationships under different day‐lengths. We investigated their monogenic, digenic, and trigenic effects on target gene regulation and PS variation.We found that Hd1 and Ghd7 have the primary functions for promoting and repressing flowering, respectively, regardless of day‐length. However, under LD conditions, Hd1 promotes Ghd7 expression and is recruited by Ghd7 and/or DTH8 to form repressive complexes that collaboratively suppress the Ehd1‐Hd3a/RFT1 pathway to block heading, but under SD conditions Hd1 competes with the complexes to promote Hd3a/RFT1 expression, playing a tradeoff relationship with PS flowering. Natural allelic variations of Hd1, Ghd7 and DTH8 in rice populations have resulted in various PS performances.Our findings reveal that rice PS flowering is controlled by crosstalk of two modules – Hd1–Hd3a/RFT1 in SD conditions and (Hd1/Ghd7/DTH8)–Ehd1–Hd3a/RFT1 in LD conditions – and the divergences of these genes provide the basis for rice adaptation to broad regions.

Oryza rufipogon Griff. is a wild progenitor of the Asian cultivated rice Oryza sativa. To better understand the genomic diversity of the wild rice, high-quality reference genomes of O. rufipogon populations are needed, which also facilitate utilization of the wild genetic resources in rice breeding. In this study, we generated a chromosome-level genome assembly of O. rufipogon using a combination of short-read sequencing, single-molecule sequencing, BioNano and Hi-C platforms. The genome sequence (399.8 Mb) was assembled into 46 scaffolds on the 12 chromosomes, with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb, respectively. The genome contains 36,520 protein-coding genes, and 49.37% of the genome consists of repetitive elements. The genome has strong synteny with those of the O. sativa subspecies indica and japonica, but containing some large structural variations. Evolutionary analysis unveiled the polyphyletic origins of O. sativa, in which the japonica and indica genome formations involved different divergent O. rufipogon (including O. nivara) lineages, accompanied by introgression of genomic regions between japonica and indica. This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O. sativa subspecies, and valuable information for basic research and rice breeding.

Rice mitochondrial aldehyde dehydrogenase OsALDH2b contributes to tapetal degradation, which is essential for male fertility., Timely degradation of anther tapetal cells is a prerequisite for normal pollen development in flowering plants. Although several genes involved in tapetum development have been identified, the molecular basis of tapetum degeneration regulation remains poorly understood. In this study, we identified and characterized the nucleus-encoded, conserved mitochondrial aldehyde dehydrogenase OsALDH2b as a key regulator of tapetum degeneration in rice (Oryza sativa). OsALDH2b was highly expressed in anthers from meiosis to the early microspore stage. Mutation of OsALDH2b resulted in excess malonaldehyde accumulation and earlier programmed cell death in the tapetum, leading to premature tapetum degeneration and abnormal microspore development. These results demonstrate that OsALDH2b negatively regulates tapetal programmed cell death and is required for male reproductive development, providing insights into the regulation of tapetum development in plants.

Flowers are the core reproductive organ of plants, and flowering is essential for cross-pollination. Diurnal flower-opening time is thus a key trait influencing reproductive isolation, hybrid breeding, and thermostability in plants. However, the molecular mechanisms controlling this trait remain unknown. Here, we report that rice Diurnal Flower Opening Time 1 (DFOT1) modulates pectin methylesterase (PME) activity to regulate pectin methylesterification levels of the lodicule cell walls, which affect lodicule swelling to control diurnal flower-opening time. DFOT1 is specifically expressed in the lodicules, and its expression gradually increases with the approach to flowering but decreases with flowering. Importantly, a knockout of DFOT1 showed earlier diurnal flower opening. We demonstrate that DFOT1 interacts directly with multiple PMEs to promote their activity. Knockout of PME40 also resulted in early diurnal flower opening, whereas overexpression of PME42 delayed diurnal flower opening. Lower PME activity was observed to be associated with higher levels of pectin methylesterification and the softening of cell walls in lodicules, which contribute to the absorption of water by lodicules and cause them to swell, thus promoting early diurnal flower opening. Higher PME activity had the opposite effect. Collectively, our work uncovers a molecular mechanism underlying the regulation of diurnal flower-opening time in rice, which would help reduce the costs of hybrid breeding and improve the heat tolerance of flowering plants by avoiding higher temperatures at anthesis.

Despite continuous improvements, it is difficult to efficiently amplify large sequences from complex templates using current PCR methods. Here, we developed a suppression thermo-interlaced (STI) PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools. This method uses site-specific primers containing a common 5' tag to generate a stem-loop structure, thereby repressing the amplification of smaller non-specific products through PCR suppression (PS). However, large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed. Furthermore, this method uses nested thermo-interlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution. The combination of these two factors in STI PCR produces a multiplier effect, markedly increasing specificity and amplification capacity. We also developed a webtool, calGC, for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR. Using this method, we stably amplified very long genomic fragments (up to 38 kb) from plants and human and greatly increased the length of de novo DNA synthesis, which has many applications such as cloning, expression, and targeted genomic sequencing. Our method greatly extends PCR capacity and has great potential for use in biological fields.

Plant height is an important agronomic trait for lodging resistance and yield. Here, we report a new plant-height-related gene, OsUBR7 in rice (Oryza sativa L.); knockout of OsUBR7 caused fewer cells in internodes, resulting in a semi-dwarf phenotype. OsUBR7 encodes a putative E3 ligase containing a plant homeodomain finger and a ubiquitin protein ligase E3 component N-recognin 7 (UBR7) domain. OsUBR7 interacts with histones and monoubiquitinates H2B (H2Bub1) at lysine148 in coordination with the E2 conjugase OsUBC18. OsUBR7 mediates H2Bub1 at a number of chromatin loci for the normal expression of target genes, including cell-cycle-related and pleiotropic genes, consistent with the observation that cell-cycle progression was suppressed in the osubr7 mutant owing to reductions in H2Bub1 and expression levels at these loci. The genetic divergence of OsUBR7 alleles among japonica and indica cultivars affects their transcriptional activity, and these alleles may have undergone selection during rice domestication. Overall, our results reveal a novel mechanism that mediates H2Bub1 in plants, and UBR7 orthologs could be utilized as an untapped epigenetic resource for crop improvement.

Soybean, a typical short-day crop, is sensitive to photoperiod, which is a major limiting factor defining its north-to-south cultivation range. The long-juvenile (LJ) trait is controlled primarily by the J locus which has been used for decades by soybean breeders to delay flowering and improve grain yield in tropical regions. The J gene encodes an ortholog of the Arabidopsis Evening Complex (EC) component EARLY FLOWERING 3 (ELF3). To identify modifiers of J, we conducted a forward genetic screen and isolated a mutant (eoj57) that in combination with j has longer flowering delay compared with j single mutant plants. Map-based cloning and genome re-sequencing identified eoj57 (designated as GmLUX2) as an ortholog of the Arabidopsis EC component LUX ARRHYTHMO (LUX). To validate that GmLUX2 is a modifier of J, we used trans-complementation and identified a natural variant allele with a similar phenotype. We also show that GmLUX2 physically interacts with GmELF3a/b and binds DNA, whereas the mutant and natural variant are attenuated in both activities. Transcriptome analysis shows that the GmLUX2-GmELF3a complex co-regulates the expression of several circadian clock-associated genes and directly represses E1 expression. These results provide mechanistic insight into how GmLUX2-GmELF3 controls flowering time via synergistic regulation of gene expression. These novel insights expand our understanding of the regulation of the EC complex, and facilitate the development of soybean varieties adapted for growth at lower latitudes.

Genome editing with CRISPR/Cas9 has become an effective tool for gene functionality dissection and crop improvement. Accordingly, we have developed a CRISPR/Cas9 vector system for robust multi-gene editing in plants. Our inventory includes six versatile binary vectors and 12 intermediary sgRNA vectors with different U3 and U6 promoters, to achieve high efficient multi-gene editing, under specific antibiotic selections in both monocot and dicot plants. Additionally, we also have developed an integrated web-based software toolkit, CRISPR-GE, to facilitate the utilization CRISPR/Cas9 in plants. In this protocol, we provide detailed procedures for using the CRISPR/Cas9 system, including target site selection, construction of CRISPR/Cas9 binary vectors, Sanger-sequencing-based genotyping of mutant sites, and troubleshooting for common problems.

Use of the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) and Cpf1 systems in plants (Ma et al., 2016; Wang et al., 2017) involves many steps, including the selection of appropriate specific target site(s) that should have no highly homologous sequences as the potential off-target sites in the genome, the design and synthesis of oligonucleotides involving the target sequences, the preparation of expression cassette(s) for the target single guide RNAs (sgRNAs) that provide target sequence specificity, the construction of plant-transformation/expression vector(s), and the transformation of plants, followed by the detection and determination of the targeted mutations in the transgenic plants.

In many diploid organisms, the majority mutations induced by clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing are non- chimeric, including biallelic, homozygous, and heterozygous mutations. Direct Sanger sequencing of the PCR amplicons containing non-homozygous mutations superimposes sequencing chromatograms, displaying overlapping peaks beginning from the mutation sites. In this chapter we describe the degenerate sequence decoding (DSD) strategy and its automatic web-based tool, DSDecodeM, for decoding the Sanger sequencing chromatograms from different types of targeted mutations. DSDecodeM, as a convenient and versatile tool, can considerably facilitate the genotyping work of CRISPR-induced mutants.

In many diploid organisms, the majority mutations induced by clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing are non- chimeric, including biallelic, homozygous, and heterozygous mutations. Direct Sanger sequencing of the PCR amplicons containing non-homozygous mutations superimposes sequencing chromatograms, displaying overlapping peaks beginning from the mutation sites. In this chapter we describe the degenerate sequence decoding (DSD) strategy and its automatic web-based tool, DSDecodeM, for decoding the Sanger sequencing chromatograms from different types of targeted mutations. DSDecodeM, as a convenient and versatile tool, can considerably facilitate the genotyping work of CRISPR-induced mutants.

New gene origination is a major source of genomic innovations that confer phenotypic changes and biological diversity. Generation of new mitochondrial genes in plants may cause cytoplasmic male sterility (CMS), which can promote outcrossing and increase fitness. However, how mitochondrial genes originate and evolve in structure and function remains unclear. The rice Wild Abortive type of CMS is conferred by the mitochondrial gene WA352c (previously named WA352) and has been widely exploited in hybrid rice breeding. Here, we reconstruct the evolutionary trajectory of WA352c by the identification and analyses of 11 mitochondrial genomic recombinant structures related to WA352c in wild and cultivated rice. We deduce that these structures arose through multiple rearrangements among conserved mitochondrial sequences in the mitochondrial genome of the wild rice Oryza rufipogon, coupled with substoichiometric shifting and sequence variation. We identify two expressed but nonfunctional protogenes among these structures, and show that they could evolve into functional CMS genes via sequence variations that could relieve the self-inhibitory potential of the proteins. These sequence changes would endow the proteins the ability to interact with the nucleus-encoded mitochondrial protein COX11, resulting in premature programmed cell death in the anther tapetum and male sterility. Furthermore, we show that the sequences that encode the COX11-interaction domains in these WA352c-related genes have experienced purifying selection during evolution. We propose a model for the formation and evolution of new CMS genes via a “multi-recombination/protogene formation/functionalization” mechanism involving gradual variations in the structure, sequence, copy number, and function.

Phenylalanine ammonia-lyase (PAL) is the first enzyme involved in the phenylpropanoid pathway and plays important roles in the secondary metabolisms, development and defense of plants. To study the molecular function of PAL in anthocyanin synthesis of Coleus (Solenostemon scutellarioides (L.) Codd), a Coleus PAL gene designated as SsPAL1 was cloned and characterized using a degenerate oligonucleotide primer PCR and RACE method. The full-length SsPAL1 was 2450 bp in size and consisted of one intron and two exons encoding a polypeptide of 711 amino acids. The deduced SsPAL1 protein showed high identities and structural similarities with other functional plant PAL proteins. A series of putative cis-acting elements involved in transcriptional regulation, light and stress responsiveness were found in the upstream regulatory sequence of SsPAL1. Transcription pattern analysis indicated that SsPAL1 was constitutively expressed in all tissues examined and was enhanced by light and different abiotic factors. The recombinant SsPAL1 protein exhibited high PAL activity, at optimal conditions of 60 °C and pH 8.2. Although the levels of total PAL activity and total anthocyanin concentration have a similar variation trend in different Coleus cultivars, there was no significant correlation between them (r = 0.7529, p &gt, 0.1), suggesting that PAL was not the rate-limiting enzyme for the downstream anthocyanin biosynthetic branch in Coleus. This study enables us to further understand the role of SsPAL1 in the phenylpropanoid (flavonoids, anthocyanins) biosynthesis in Coleus at the molecular level.

Recently, genome editing technologies have shown great potential in plants. The newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a new generation of genome editing tool rapidly replacing the earlier zinc finger nucleases and transcription activator-like effector nucleases systems. Indeed, due to its advantages of simplicity and high efficiency, the CRISPR/Cas9-based genome editing system is becoming a powerful tool in plant science research. Here, we introduce the technical features of the plant CRISPR/Cas9-based genome editing system and its applications in plant functional genomics studies and genetic improvement.

Hybrids between divergent populations commonly show hybrid sterility; this reproductive barrier hinders hybrid breeding of the japonica and indica rice (Oryza sativa L.) subspecies. Here we show that structural changes and copy number variation at the Sc locus confer japonica–indica hybrid male sterility. The japonica allele, Sc-j, contains a pollen-essential gene encoding a DUF1618-domain protein; the indica allele, Sc-i, contains two or three tandem-duplicated ~ 28-kb segments, each carrying an Sc-j-homolog with a distinct promoter. In Sc-j/Sc-i hybrids, the high-expression of Sc-i in sporophytic cells causes suppression of Sc-j expression in pollen and selective abortion of Sc-j-pollen, leading to transmission ratio distortion. Knocking out one or two of the three Sc-i copies by CRISPR/Cas9 rescues Sc-j expression and male fertility. Our results reveal the gene dosage-dependent allelic suppression as a mechanism of hybrid incompatibility, and provide an effective approach to overcome the reproductive barrier for hybrid breeding., A reproductive barrier between japonica and indica rice subspecies hinders hybrid breeding. Here, the authors reveal that structural and copy number variations of a pollen-essential gene, which encodes a DUF1618-domain protein, result in allelic suppression and subsequently cause hybrid male sterility.

Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice ( Oryza sativa L.), could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes (two regulatory genes from maize and six structural genes from Coleus) driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm "Purple Endosperm Rice" (called "Zijingmi" in Chinese), which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation (or enhancement) of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.

Recently, genome editing technologies have shown great potential in plants. The newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a new generation of genome editing tool rapidly replacing the earlier zinc finger nucleases and transcription activator-like effector nucleases systems. Indeed, due to its advantages of simplicity and high efficiency, the CRISPR/Cas9-based genome editing system is becoming a powerful tool in plant science research. Here, we introduce the technical features of the plant CRISPR/Cas9-based genome editing system and its applications in plant functional genomics studies and genetic improvement.

The transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing systems have greatly improved the efficiency for generating targeted mutations in various organisms including plants (Li et al., 2012; Cong et al., 2013; Li et al., 2013; Feng et al., 2014; Ma et al., 2015b; Zhang et al., 2014, 2015). In some plant species, the majority of mutations induced by TALENs and CRISPR/Cas9 systems are in uniform biallelic and heterozygous status in the first transgenic generation, although in some other plant species, chimeric mutations (with three or more allelic edited events within a single individual) may frequently occur (Li et al., 2013; Feng et al., 2014; Zhang et al., 2014, 2015; Ma et al., 2015b).