Genome editing for plant synthetic metabolic engineering and developmental regulation.

Plant metabolism and development are a reflection of the orderly expression of genetic information intertwined with the environment interactions. Genome editing is the cornerstone for scientists to modify endogenous genes or introduce exogenous functional genes and metabolic pathways, holding immense potential applications in molecular breeding and biosynthesis. Over the course of nearly a decade of development, genome editing has advanced significantly beyond the simple cutting of double-stranded DNA, now enabling precise base and fragment replacements, regulation of gene expression and translation, as well as epigenetic modifications. However, the utilization of genome editing in plant synthetic metabolic engineering and developmental regulation remains exploratory. Here, we provide an introduction and a comprehensive overview of the editing attributes associated with various CRISPR/Cas tools, along with diverse strategies for the meticulous control of plant metabolic pathways and developments. Furthermore, we discuss the limitations of current approaches and future prospects for genome editing-driven plant breeding.

Progress and prospect: Biosynthesis of plant natural products based on plant chassis.

Plant-derived natural products are a specific class of active substances with numerous applications in the medical, energy, and industrial fields. Many of these substances are in high demand and have become the fundamental materials for various purposes. Recently, the use of synthetic biology to produce plant-derived natural products has become a significant trend. Plant chassis, in particular, offer unique advantages over microbial chassis in terms of cell structure, product affinity, safety, and storage. The development of the plant hairy root tissue culture system has accelerated the commercialization and industrialization of synthetic biology in the production of plant-derived natural products. This paper will present recent progress in the synthesis of various plant natural products using plant chassis, organized by the types of different structures. Additionally, we will summarize the four primary types of plant chassis used for synthesizing natural products from plant sources and review the enabling technologies that have contributed to the development of synthetic biology in recent years. Finally, we will present the role of isolated and combined use of different optimization strategies in breaking the upper limit of natural product production in plant chassis. This review aims to provide practical references for synthetic biologists and highlight the great commercial potential of plant chassis biosynthesis, such as hairy roots.

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.

BEtarget: A versatile web-based tool to design guide RNAs for base editing in plants.

CRISPR-dependent base editors enable direct nucleotide conversion without the introduction of double-strand DNA break or donor DNA template, thus expanding the CRISPR toolbox for genetic manipulation. However, designing guide RNAs (gRNAs) for base editors to enable gene correction or inactivation is more complicated than using the CRISPR system for gene disruption. Here, we present a user-friendly web tool named BEtarget dedicated to the design of gRNA for base editing. It is currently supported by 46 plant reference genomes and 5 genomes of non-plant model organisms. BEtarget supports the design of gRNAs with different types of protospacer adjacent motifs (PAM) and integrates various functions, including automatic identification of open reading frame, prediction of potential off-target sites, annotation of codon change, and assessment of gRNA quality. Moreover, the program provides an interactive interface for users to selectively display information about the desired target sites. In brief, we have developed a flexible and versatile web-based tool to simplify complications associated with the design of base editing technology. BEtarget is freely accessible at https://skl.scau.edu.cn/betarget/.

Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools.

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.

Efficient assembly of long DNA fragments and multiple genes with improved nickase-based cloning and Cre/loxP recombination.

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.

Molecular farming using transgenic rice endosperm.

Plant expression platforms are low-cost, scalable, safe, and environmentally friendly systems for the production of recombinant proteins and bioactive metabolites. Rice (Oryza sativa L.) endosperm is an ideal bioreactor for the production and storage of high-value active substances, including pharmaceutical proteins, oral vaccines, vitamins, and nutraceuticals such as flavonoids and carotenoids. Here, we explore the use of molecular farming from producing medicines to developing functional food crops (biofortification). We review recent progress in producing pharmaceutical proteins and bioactive substances in rice endosperm and compare this platform with other plant expression systems. We describe how rice endosperm could be modified to design metabolic pathways and express and store stable products and discuss the factors restricting the commercialization of transgenic rice products and future prospects.

All-in-one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants.

Fluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein-protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co-transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all-in-one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single-tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high-throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS-guided GA-mediated AioFFP vector toolkit is cost-effective, easy to use and will promote functional genomics research in plants.

An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

PhieABEs: a PAM-less/free high-efficiency adenine base editor toolbox with wide target scope in plants.

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.

OsEDM2L mediates m6 A of EAT1 transcript for proper alternative splicing and polyadenylation regulating rice tapetal degradation.

N6 -methyladenosine (m6 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 N6 -adenine methyltransferase-like domain-containing protein ENHANCED DOWNY MILDEW 2-LIKE (OsEDM2L) is essential for rice (Oryza sativa L.) anther development. The osedm2l knockout mutant showed delayed tapetal programmed cell death (PCD) and defective pollen development. OsEDM2L interacts with the transcription factors basic helix-loop-helix 142 and TAPETUM DEGENERATION RETARDATION to regulate the expression of ETERNAL TAPETUM 1 (EAT1), a positive regulator of tapetal PCD. Mutation of OsEDM2L altered the transcriptomic m6 A landscape, and caused a distinct m6 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, OsEDM2L is indispensable for proper messenger RNA m6 A modification in rice anther development.

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 -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.

TransGene Stacking II Vector System for Plant Metabolic Engineering and Synthetic Biology.

Efficient stacking of multiple genes is a critical element in metabolic engineering of complex pathways, synthetic biology, and genetic improvement of complex agronomic traits in plants. Here we present a high-efficiency multigene assembly and transformation vector system, TransGene Stacking II (TGS II), for these purposes. The operation process is described in detail, and the successful operation mainly depends on effective reagents, special Escherichia coli strains, and basic molecular biological means without other specific equipments.

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 carotenoid 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 chromoplast biogenesis, dramatically enhances provitamin A and total carotenoid content and stability. Up to 65- and 10-fold increases of ß-carotene and total carotenoids, respectively, with provitamin A carotenoids composing over 63% were observed in the seeds containing OR His and PSY. Co-expression of Homogentisate geranylgeranyl transferase (HGGT) with OR His and PSY further increases carotenoid accumulation and stability during seed maturation and storage. Moreover, knocking-out of ß-carotene hydroxylase 2 (BCH2) by CRISPR/Cas9 not only potentially facilitates ß-carotene accumulation but also minimizes the negative effect of carotenoid over production on seed germination. Our findings provide new insights into various processes on carotenoid accumulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis, turnover, and stable storage for carotenoid biofortification in crop seeds. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00046-1.

Prospects and progress on crocin biosynthetic pathway and metabolic engineering.

Crocins are a group of highly valuable apocarotenoid-derived pigments mainly produced in Crocus sativus stigmas and Gardenia jasminoides fruits, which display great pharmacological activities for human health, such as anticancer, reducing the risk of atherosclerosis, and preventing Alzheimer's disease. However, traditional sources of crocins are no longer sufficient to meet current demands. The recent clarification of the crocin biosynthetic pathway opens up the possibility of large-scale production of crocins by synthetic metabolic engineering methods. In this review, we mainly introduce the crocin biosynthetic pathway, subcellular route, related key enzymes, and its synthetic metabolic engineering, as well as its challenges and prospects, with a view to providing useful references for further studies on the synthetic metabolic engineering of crocins.

Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.

Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.