Overexpression of maize GOLDEN2 in rice and maize calli improves regeneration by activating chloroplast development

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.

基于CRISPR编辑系统的DNA片段删除技术

基于CRISPR/Cas9系统的基因组编辑技术已成为基因功能研究和遗传修饰的重要工具。在引导RNA的引导下,Cas9蛋白对基因组靶位点进行精准切割产生DNA双链断裂(DSB),借助细胞内的DSB修复机制,可实现基因组靶位点碱基的缺失、插入或者替换,甚至发生片段删除。该文介绍了基于CRISPR/Cas9基因组编辑系统的DSB微同源末端连接修复方式(MMEJ)提高基因组DNA片段删除效率的方法,主要从靶点设计和突变检测方面展开详细描述。

A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants

CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high- efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/ Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edi- ted 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homol- ogous end-joining mechanism followed by homologous recombination-based repair. We also obtained uni- form biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mu- tations in To rice and T1Arabidopsis plants by simultaneous targeting of multiple （up to eight） members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

XA23 Is an Executor R Protein and Confers Broad-Spectrum Disease Resistance in Rice

The majority of plant disease resistance （R） genes encode proteins that share common structural features. However, the transcription activator-like effector （TALE）-associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from wild rice （Oryza rufipogon） that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae （Xoo）. Xa23 encodes a 113 amino acid protein that shares 50% identity with the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike XalO, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23 but differs in promoter region by lacking the TALE binding element （EBE） for AvrXa23. XA23 can trigger a strong hypersensitive response in rice, tobacco, and tomato. Our results provide the first evidence that plant genomes have an executor R gene family of which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in the pathogen.

From Golden Rice to aSTARice:Bioengineering Astaxanthin Biosynthesis in Rice Endosperm

Carotenoids are important phytonutrients with antioxidant properties,and are widely used in foods and feedstuffs as Supplements.Astaxanthin,a red-colored ketocarotenoid,has strong antioxidant activity and thus can benefit human health.However,astaxanthin is not produced in most higher plants.Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes,sZmPSY1,sPaCrtl,sCrBKT,and sHpBHY,which encode the enzymes phytoene synthase,phytoene desaturase,β-carotene ketolase,and β-carotene hydroxylase,respectively.Transgneic overexpression of two (sZmPSY1 and sPaCrtl),three (sZmPSY1,sPaCrtl and sCrBKT),and all these four genes driven by rice endosperm-specific promoters established the Carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm,from the yellow-grained β-caro- tene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice,respectively. Grains Of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity.These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in therice endosperm.This work provides a Successful example for synthetic biology in plants and biofortification in crops;the biofortified rice products generated by this study could be consumed as health-promoting foods and processed tO produce dietary supplements.

Development of ＂Purple Endosperm Rice＂ by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System

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.

DRISPR/Cas9 Platforms for Genome Editing in Plants： Developments and Applications

The clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein9 （Cas9） genome editing system （CRISPR/Casg） is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adap- tion of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/ Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, fac- tors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.

Targeted Genome Editing in Genes and cis- Regulatory Regions Improves Qualitative and Quantitative Traits in Crops

The Clustered Regularly Interspaced Short Palindromic Repeats （CRISPR）-associated protein 9 （CRISPR/Cas9）-based genomeediting system is a revolutionary technology for targeted muta- genesis in molecular biology research and genetic improvement of traits in crops （Cong et al., 2013; Ma et al., 2015, 2016）. Agronomic traits of crops are controlled by major genes and quantitative trait loci （QTL）. Therefore, the CRISPR/Cas9 system can be used to effectively and rapidly produce mutant traits by different strategies （Figure 1A-1C）. The most common application of the targeted editing system in genetic improvement is to knock out completely the functions of target genes, usually by editing site（s） in the coding sequences （CDS） to produce null-allele mutants （Figure 1A）.

A modified high-efficiency thermal asymmetric interlaced PCR method for amplifying long unknown flanking sequences

Rapid and efficient isolation of unknown flanking DNA sequences adjacent to known regions is important for molecular biology research.For this purpose,several PCR-based methods have been reported,including inverse PCR(Uchiyama and Watanabe,2006),ligation-mediated PCR(Yan et al.,2003;Ballester et al.,2005;Wang et al.,2007;Trinh et al.,2012)and randomly primed PCR(Liu and Whittier,1995;Liu et al.,1995;Antal et al.

PhieCBEs:Plant High-Efficiency Cytidine Base Editors with Expanded Target Range

Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated genome editing can efficiently produce gene-knockout mutants.On the other hand,CRISPR/Cas-derived base editors offer the ability to induce precise nucleotide substitutions(Komor et al.,2016).Cytidine base editors(CBEs)consist of a cytidine deaminase fused with a Cas9-nickase variant(Cas9n,with a D10A substitu-tion)and can achieve site-specific C-to-T substitution.Similarly,adenine base editors use an adenine deaminase forA-to-G substi-tution.These systems have been used in various organisms(Mishra et al.,2019).However,the Cas9 complex requires target sites containing NGG protospacer adjacent motifs(PAMs),thus restricting selection of potential targets.A number of CBEs have been developed using Cas9 variants(mostly Cas9n),cytidine deaminases(such as rAPOBEC1 and PmCDA1),and uracil glycosylase inhibitor(UGI)domains.These CBEs of the first generation(BE1,rAPOBEC1-dCas9),second generation(BE2,rAPOBEC1-dCas9-UGI),and third generation(BE3,rAPOBEC1-Cas9n-UGI)have moderate editing efficiencies in mammalians(Komor etal.,2016).

An effective strategy to establish a male sterility mutant mini-library by CRISPR/Cas9-mediated knockout of anther-specific genes in rice

Male sterility(MS),characterized by functional defects in male organs or gametes,is an important agronomic trait for hybrid seed production,especially for self-pollinated crops such as rice(Oryza sativa L)(Chen and Liu,2014).Spontaneous MS mutants are rare and difficult to maintain in nature,thus limiting basicresearch and breeding.Artificial mutants are typically generated by physical,chemical,or biological mutagenesis(Wei et al,2013).Recently developed genome editing systems such as CRISPR/Cas9 allow efficient and timesaving knockout of endogenous genes at specific sites(Smith et al,2000;Moscou and Bogdanove,2009;Gasiunas et al.,2012).

A novel CCCH-type zinc finger protein SAW1 activates OsGA20ox3 to regulate gibberellin homeostasis and anther development in rice

Male sterility is a prerequisite for hybrid seed production.The phytohormone gibberellin(GA)is in-volved in regulating male reproductive development,but the mechanism underlying GA homeostasis in anther development remains less understood.Here,we report the isolation and characterization of a new positive regulator of GA homeostasis,swollen anther wall 1(SAW1),for anther development in rice(Oryza sativa L.).Rice plants carrying the recessive mutant allele saw1 produces abnormal anthers with swollen anther wall and aborted pollen.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRIPSR-associated protein 9-mediated knockout of SAW1 in rice generated similar male sterile plants.SAW1 encodes a novel nucleus-localizing CCCH-tandem zinc finger protein,and this protein could directly bind to the promoter region of the GA synthesis gene OsGA20ox3 to induce its anther-specific expression.In the saw1 anther,the significantly decreased OsGA20ox3 expression resulted in lower bio-active GA content,which in turn caused the lower expression of the GA-inducible anther-regulator gene OsGAMYB.Thus,our results disclose the mechanism of the SAW1-GA20ox3-GAMYB pathway in controlling rice anther development,and provide a new target gene for the rapid generation of male sterile lines by genome editing for hybrid breeding.

The Intronic cis Element SE1 Recruits trans-Acting Repressor Complexes to Repress the Expression of ELONGATED UPPERMOST INTERNODE1 in Rice

Plant height has a major effect on grain yield in crops such as rice （Oryza sativa）, and the hormone gibberellic acid （GA） regulates many developmental processes that feed into plant height. Rice ELONGATED UPPERMOST INTERNODE1 （Euil） encodes a GA-deactivating enzyme governing elongation of the uppermost internode. The expression of Euil is finely tuned, thereby maintaining homeostasis of endogenous bioactive GA and producing plants of normal plant height. Here, we identified a dominant dwarf mutant, dEuil, caused by the deletion of an RY motif-containing cis-silencing element （SE1） in the intron of Euil. Detailed genetic and molecular analysis of SE1 revealed that this intronic cis element recruits at least one trans-acting repressor complex, containing the B3 repressors OsVAL2 and OsGD1, the SAP18 corepressor, and the histone deacetylase OsHDA710, to negatively regulate the expression of Euil. This com- plex generates closed chromatin at Euil, suppressing Euil expression and modulating GA homeostasis. Loss of SE1 or dysfunction of the complex components impairs histone deacetylation and H3K27me3 methylation of Euil chromatin, thereby increasing Euil transcription and decreasing bioactive GA, producing dwarfism in rice. Together, our results reveal a novel silencing mechanism in which the intronic cis element SE1 negatively regulates Euil expression via repressor complexes that modulate histone deacetylation and/or methylation.

Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants

Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA(sgRNA)incorporating a target sequence(Li et al.,2019).A Cas9/sgRNA complex recognizes its target site in the genome,resulting in a mutation at that site.

Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds

Staple grains with low levels of provitamin A carotenoids contribute to the global prevalence of vitamin A deficiency and therefore are the main targets for provitamin A biofortification.However,carotenoid stability during both seed maturation and postharvest storage is a serious concern for the full benefits of carotenoid biofortified grains.In this study,we utilized Arabidopsis as a model to establish car-otenoid biofortification strategies in seeds.We discovered that manipulation of carotenoid biosynthetic activity by seed-specific expression of Phytoene synthase(PSY)increases both provitamin A and total carotenoid levels but the increased carotenoids are prone to degradation during seed maturation and storage,consistent with previous studies of provitamin A biofortified grains.In contrast,stacking with Orange(OR^(His)),a gene that initiates chromopl ast biogenesis,dramatically enhances provitamin A and total carotenoid content and stability.Up to 65-and 10-fold increases of β-carotene and total car-otenoids,res pectively,with provitamin A carotenoids composing over 63%were observed in the seeds containing OR^(His) and PSY.Co-expression of Homogen tisate geranylgeranyl transferase(HGGT)with OR^(His) and PSY further increases carotenoid accumulation and stability during seed maturation and storage.Moreover,knocking-out of B-carotene hydroxylase 2(BCH2)by CRISPR/Cas9 not only potentially facilitates β-carotene accumulation but also minimizes the negative effect of carotenoid over production on seed germi nation.Our findings provide new insights into various processes on carotenoid accu-mulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis,turnover,and stable storage for carotenoid biofortification in crop seeds.

MMEJ-KO:a web tool for designing paired CRISPR guide RNAs for microhomology-mediated end joining fragment deletion

Dear Editor,Clustered regularly interspaced short palindromic repeats associated protein(CRISPR/Cas)systems are convenient and versatile tools for genome editing.Directed by a guide RNA(gRNA),the Cas nuclease produces double-strand break(DSB)at the target site,generally resulting in small base insertion/deletion mutations by the non-homologous end joining(NHEJ)DNA repair.

OsEDM2L mediates m^(6)A of EAT1 transcript for proper alternative splicing and polyadenylation regulating rice tapetal degradation

N^(6)-methyladenosine(m^(6)A) modification affects the post-transcriptional regulation of eukaryotic gene expression, but the underlying mechanisms and their effects in plants remain largely unknown. Here,we report that the N^(6)-adenine methyltransferase-like domain-containing protein ENHANCED DOWNY MILDEW 2-LIKE(OsEDM2 L) is essential for rice(Oryza sativa L.) anther development. The osedm2 l knockout mutant showed delayed tapetal programmed cell death(PCD) and defective pollen development. OsEDM2 L interacts with the transcription factors basic helix-loop-helix 142 and TAPETUMDEGENERATIONRETARDATIONto regulate the expression of ETERNAL TAPETUM 1(EAT1), a positive regulator of tapetal PCD. Mutation of OsEDM2 L altered the transcriptomic m^(6)A landscape, and caused a distinct m^(6)A modification of the EAT1 transcript leading to dysregulation of its alternative splicing and polyadenylation, followed by suppression of the EAT1 target genes OsAP25 and OsAP37 for tapetal PCD. Therefore, OsEDM2 L is indispensable for proper messenger RNA m^(6)A modification in rice anther development.

Plant Synthetic Metabolic Engineering for Enhancing Crop Nutritional Quality

Nutrient deficiencies in crops are a serious threat to human health,especially for populations in poor areas.To overcome this problem,the development of crops with nutrient-enhanced traits is imperative.Biofortification of crops to improve nutritional quality helps combat nutrient deficiencies by increasing the levels of specific nutrient components.Compared with agronomic practices and conventional plant breeding,plant metabolic engineering and synthetic biology strategies are more effective and accurate in synthesizing specific micronutrients,phytonutrients,and/or bioactive components in crops.In this review,we discuss recent progress in the field of plant synthetic metabolic engineering,specifically in terms of research strategies of multigene stacking tools and engineering complex metabolic pathways,with a focus on improving traits related to micronutrients,phytonutrients,and bioactive components.Advances and innovations in plant synthetic metabolic engineering would facilitate the development of nutrient-enriched crops to meet the nutritional needs of humans.

The ScCas9^(++) variant expands the CRISPR toolbox for genome editing in plants

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)-C_(17)) 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.