Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease‐susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9‐targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1^G allele and one copy of the CsLOB1^? allele. The promoter of both alleles contains the effector binding element (EBE_PthA4), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBE_PthA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%–64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBE_PthA4 modifications were identified from 38 mutant plants. Four mutation lines (S2‐5, S2‐6, S2‐12 and S5‐13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2‐6 and S5‐13 lines. Promoter editing of CsLOB1^G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE_PthA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9‐mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker‐resistant citrus cultivars.     

Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.     

快速构建多重sgRNA载体利用CRISPR/Cas9技术敲除拟南芥IAA2基因

IAA2(Indole Acetic Acid 2)是拟南芥Aux/IAA生长素响应基因大家族中的一员,目前还没有它的突变体的报道,阻碍了对其功能和作用机制的深入研究。在CRISPR/Cas9基因组编辑技术中,1个sgRNA只能靶向基因的1个位点,有时基因敲除的效率并不高。为了提高敲除效率,本文在Golden-Gate克隆技术的基础上,通过两轮PCR扩增,将每3个sgRNA串联到同1个入门载体中,再将入门载体与含Cas9表达框的目标载体LR反应,获得最终的表达载体。结果表明,设计的6个sgRNA有4个发挥了作用,产生了碱基插入突变和大片段缺失突变等多种可遗传的突变。与单个sgRNA相比,多重sgRNA的基因敲除效率高、种系突变多;与其他构建多重sgRNA载体的方法相比,本方法具有快速、高效等优点。本文所得到的5个突变体为后续的IAA2功能研究提供了良好的材料。     

CRISPR/Cas9基因组编辑技术在HIV-1感染治疗中的应用进展

基因治疗是将外源正常基因通过一定方式导入人体靶细胞以纠正或补偿因基因缺陷和异常引起的疾病,从而达到治疗目的。因此,基因治疗的技术方法在研究持续感染HIV-1或潜伏感染HIV-1原病毒患者的治疗中具有重大的现实意义。目前,现有的基因治疗方法存在识别靶向位点有限及脱靶几率大等主要问题。最新研究表明来源于细菌和古菌的规律间隔成簇短回文重复序列及其相关核酸酶9系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), CRISPR/Cas9]已被成功改造成基因组定点编辑工具。因此,如何利用CRISPR/Cas9系统实现对HIV-1病毒基因组进行高效靶向修饰,从而达到治疗HIV-1感染病患的目的已经成为当前研究的热点。本文参考最新国内外研究成果,重点介绍了 CRISPR/Cas9基因组编辑技术在HIV-1感染疾病治疗中的应用,主要包括CCR5基因编辑、清除HIV-1原病毒以及活化HIV-1原病毒,以期为HIV-1感染疾病的预防与治疗提供重要研究参考。     

Multiplexed CRISPR/Cas9‐mediated metabolic engineering of γ‐aminobutyric acid levels in Solanum lycopersicum

In recent years, the type II CRISPR system has become a widely used and robust technique to implement site‐directed mutagenesis in a variety of species including model and crop plants. However, few studies manipulated metabolic pathways in plants using the CRISPR system. Here, we introduced the pYLCRISPR/Cas9 system with one or two single‐site guide RNAs to target the tomato phytoene desaturase gene. An obvious albino phenotype was observed in T0 regenerated plants, and more than 61% of the desired target sites were edited. Furthermore, we manipulated the γ‐aminobutyric acid (GABA) shunt in tomatoes using a multiplex pYLCRISPR/Cas9 system that targeted five key genes. Fifty‐three genome‐edited plants were obtained following single plant transformation, and these samples represented single to quadruple mutants. The GABA accumulation in both the leaves and fruits of genomically edited lines was significantly enhanced, and the GABA content in the leaves of quadruple mutants was 19‐fold higher than that in wild‐type plants. Our data demonstrate that the multiplex CRISPR/Cas9 system can be exploited to precisely edit tomato genomic sequences and effectively create multisite knockout mutations, which could shed new light on plant metabolic engineering regulations.     

Development of broad virus resistance in non‐transgenic cucumber using CRISPR/Cas9 technology

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

An Agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T₀ transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T₁ progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.     

Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9

Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9‐induced DNA double‐strand breaks (DSBs) in P. sojae was mediated by non‐homologous end‐joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9‐mediated cleavage of non‐mutant alleles. When donor DNA was present, homology‐directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR‐mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.     

CRISPR/Cas9‐mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean

Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens‐mediated transformation. Site‐directed mutations were observed at all targeted sites by DNA sequencing analysis. T1‐generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1‐bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58′, E116°20′). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long‐day and short‐day conditions. We identified some ‘transgene‐clean’ soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These ‘transgene‐clean’ mutants of GmFT2a may provide materials for more in‐depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction.     

Identification,characterization, and rescue of CRISPR/Cas9 generated wheat SPO11-1 mutants

Increasing crop yields through plant breeding is time consuming and laborious, with the generation of novel combinations of alleles being limited by chromosomal linkage blocks and linkage-drag. Meiotic recombination is essential to create novel genetic variation via the reshuffling of parental alleles. The exchange of genetic information between homologous chromosomes occurs at crossover (CO) sites but CO frequency is often low and unevenly distributed. This bias creates the problem of linkage-drag in recombination ‘cold’ regions, where undesirable variation remains linked to useful traits. In plants, programmed meiosis-specific DNA double-strand breaks, catalysed by the SPO11 complex, initiate the recombination pathway, although only ~5% result in the formation of COs. To study the role of SPO11-1 in wheat meiosis, and as a prelude to manipulation, we used CRISPR/Cas9 to generate edits in all three SPO11-1 homoeologues of hexaploid wheat. Characterization of progeny lines shows plants deficient in all six SPO11-1 copies fail to undergo chromosome synapsis, lack COs and are sterile. In contrast, lines carrying a single copy of any one of the three wild-type homoeologues are phenotypically indistinguishable from unedited plants both in terms of vegetative growth and fertility. However, cytogenetic analysis of the edited plants suggests that homoeologues differ in their ability to generate COs and in the dynamics of synapsis. In addition, we show that the transformation of wheat mutants carrying six edited copies of SPO11-1 with the TaSPO11-1B gene, restores synapsis, CO formation, and fertility and hence opens a route to modifying recombination in this agronomically important crop.     

CRISPR/Cas9‐mediated gene knockout in the ascidian Ciona intestinalis

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on‐target site. CRISPR/Cas9‐mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.     

CRISPR/Cas9‐mediated knockout of Ms1 enables the rapid generation of male‐sterile hexaploid wheat lines for use in hybrid seed production

The development and adoption of hybrid seed technology have led to dramatic increases in agricultural productivity. However, it has been a challenge to develop a commercially viable platform for the production of hybrid wheat (Triticum aestivum) seed due to wheat's strong inbreeding habit. Recently, a novel platform for commercial hybrid seed production was described. This hybridization platform utilizes nuclear male sterility to force outcrossing and has been applied to maize and rice. With the recent molecular identification of the wheat male fertility gene Ms1, it is now possible to extend the use of this novel hybridization platform to wheat. In this report, we used the CRISPR/Cas9 system to generate heritable, targeted mutations in Ms1. The introduction of biallelic frameshift mutations into Ms1 resulted in complete male sterility in wheat cultivars Fielder and Gladius, and several of the selected male‐sterile lines were potentially non‐transgenic. Our study demonstrates the utility of the CRISPR/Cas9 system for the rapid generation of male sterility in commercial wheat cultivars. This represents an important step towards capturing heterosis to improve wheat yields, through the production and use of hybrid seed on an industrial scale.     

Significant enhancement of fatty acid composition in seeds of the allohexaploid,Camelina sativa,using CRISPR/Cas9 gene editing

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T₂ and T₃ generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T₃ and T₄ generation Camelina seeds was associated with a combination of germ‐line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.     

Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini‐Citrus (Fortunella hindsii)

Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as ‘Mini‐Citrus’) for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig‐N50 2.2 Mb and Scaffold‐N50 5.2 Mb) for F. hindsii. In total, 32 257 protein‐coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR‐modified F. hindsii were predominantly 1‐bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T₁ mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research.     

High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10‐fold enhancements in GT frequencies have been achieved using DNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV‐based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.     

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off‐target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR‐induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5‐fold in somatic tissues and up to 100‐fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double‐stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on‐target mutagenesis in plants using CRISPR/Cas9.     

Knockout of two BnaMAX1 homologs by CRISPR/Cas9‐targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.)

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T₀ plants were edited very efficiently (56.30%–67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi‐allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T₁ and T₂ progeny. We also obtained transgene‐free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off‐target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi‐dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi‐dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.     

CRISPR/Cas9‐mediated functional recovery of the recessive rc allele to develop red rice

Red rice contains high levels of proanthocyanidins and anthocyanins, which have been recognized as health‐promoting nutrients. The red coloration of rice grains is controlled by two complementary genes, Rc and Rd. The RcRd genotype produces red pericarp in wild species Oryza rufipogon, whereas most cultivated rice varieties produce white grains resulted from a 14‐bp frame‐shift deletion in the seventh exon of the Rc gene. In the present study, we developed a CRISPR/Cas9‐mediated method to functionally restore the recessive rc allele through reverting the 14‐bp frame‐shift deletion to in‐frame mutations in which the deletions were in multiples of three bases, and successfully converted three elite white pericarp rice varieties into red ones. Rice seeds from T₁ in‐frame Rc lines were measured for proanthocyanidins and anthocyanidins, and high accumulation levels of proanthocyanidins and anthocyanidins were observed in red grains from the mutants. Moreover, there was no significant difference between wild‐type and in‐frame Rc mutants in major agronomic traits, indicating that restoration of Rc function had no negative effect on important agronomic traits in rice. Given that most white pericarp rice varieties are resulted from the 14‐bp deletion in Rc, it is conceivable that our method could be applied to most white pericarp rice varieties and would greatly accelerate the breeding of new red rice varieties with elite agronomic traits. In addition, our study demonstrates an effective approach to restore recessive frame‐shift alleles for crop improvement.