Integrative regulatory mechanisms of stomatal movements under changing climate

Journal of Integrative Plant Biology, Journal Year: 2024, Volume and Issue: 66(3), P. 368 - 393

Published: Feb. 6, 2024

Global climate change-caused drought stress, high temperatures and other extreme weather profoundly impact plant growth development, restricting sustainable crop production. To cope with various environmental stimuli, plants can optimize the opening closing of stomata to balance CO

Language: Английский

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CRISPR/Cas9-mediated genome editing techniques and new breeding strategies in cereals – current status, improvements, and perspectives

Biotechnology Advances, Journal Year: 2023, Volume and Issue: 69, P. 108248 - 108248

Published: Sept. 2, 2023

Cereal crops, including triticeae species (barley, wheat, rye), as well edible cereals (wheat, corn, rice, oat, rye, sorghum), are significant suppliers for human consumption, livestock feed, and breweries. Over the past half-century, modern varieties of cereal crops with increased yields have contributed to global food security. However, presently cultivated elite crop were developed mainly optimal environmental conditions. Thus, it has become evident that taking into account ongoing climate changes, currently a priority should be given developing new stress-tolerant cultivars. It is necessary enhance accuracy methods time required generate cultivars desired features adapt change keep up world population expansion. The CRISPR/Cas9 system been powerful versatile genome editing tool achieve desirable traits, such high-yielding, stress-tolerant, disease-resistant transgene-free lines in major cereals. Despite recent advances, application faces several challenges, amount develop lines, laboriousness, limited number genotypes may used transformation vitro regeneration. Additionally, through restricted many countries, especially Europe New Zealand, due lack flexibility GMO regulations. This review provides comprehensive update researchers interested improving using gene-editing technologies, CRISPR/Cas9. We will some critical studies on improvements their contributing factors superior technologies.

Language: Английский

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Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice

Genome biology, Journal Year: 2024, Volume and Issue: 25(1)

Published: May 21, 2024

Abstract Background High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair coexpressing a dominant-negative variant MLH1. However, this strategy has not been widely used optimization in plants, possibly because its less conspicuous effects and inconsistent performance at different sites. Results We show that direct RNAi knockdown OsMLH1 an ePE5c system increases the efficiency our most recently updated tool 1.30- to 2.11-fold stably transformed rice cells, resulting as many 85.42% homozygous mutants T 0 generation. high specificity revealed whole-genome sequencing. To overcome partial sterility induced ePE5c, conditional excision introduced remove module Cre-mediated site-specific recombination. Using simple approach enriching events, we generate 100% module-free plants increase due maintained excised whose fertility impaired. Conclusions This study provides safe reliable plant improving without disturbing development via transient MMR inhibition with excisable

Language: Английский

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Targeted deaminase‐free T‐to‐G and C‐to‐K base editing in rice by fused human uracil DNA glycosylase variants

Plant Biotechnology Journal, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 16, 2025

Base editors (BEs), a groundbreaking class of genome editing tools, enable precise single-nucleotide alterations at target genomic sites, leading to mutations that either disable or enhance gene functions, thus significantly advancing plant functional genomics research and crop enhancement (Li et al., 2023). In plants, significant advancements have been made in DNA base can directly modify adenine (A), cytosine (C) guanine (G) 2018; Zong 2017). Nevertheless, direct editor for thymine (T) remains elusive. Recently, two innovative deaminase-free glycosylase-based were developed: the gTBE T (T-to-S conversion, S = G C) gCBE C (C-to-G), enabling orthogonal modifications mammalian cells (Figure 1a; Tong 2024). These utilized fusion Cas9 nickase (nCas9) with engineered variants human uracil glycosylase (UNG), allowing excision generate apurinic/apyrimidinic (AP) sites. However, such has not developed plants date. this study, we (pTGBE) (pCKBE, K T) rice, marking substantial step forward expanding genetic manipulation capabilities plants. To establish pTSBE fused rice-codon-optimized variant UNG2Δ88-Y156A/A214T/Q259A/Y284D (mhUNGv3) (Tong 2024) nCas9 32-amino-acid linker. A bipartite nuclear localization signal peptide was UNG increase entry efficiency, resulting construct 1b). We chose ten endogenous sites targeting five genes rice test activities windows. total 400 T0 stable edited obtained Hi-TOM results showed T-to-S conversion transgenic up 78.05% efficiency 1c), but essentially no 1.85% all S1a–c). found also induced insertions deletions (InDels) frequencies ranging from 20.00% 75.32% 1c). Notably, proportion T-to-G edits (up 78.05%, averaging 39.21%) products 13.38-fold higher on average than T-to-C 3.70%, 2.93%). The is predominant type generated, purity exceeding 80% S1d), showing quite different pattern cells. cells, gTBEv3 exhibited activity efficiencies 27.26% 18.75% T-to-C, respectively Thus, designated BE as pTGBE better reflect its characteristics Furthermore, editable range positions T2–T12, T14 T18, optimal window T3–T5 highest T3 (PAM position 21–23) 1d). contrast, typically produced transversions T2-T11 T5 events included homozygous, heterozygous, biallelic chimeric-edited alleles (Table S3; Figure Homozygous conversions observed 60.00% (6/10) sgRNA maximum 30.77% (4/13) OsNRT1.1B-SG3 site, while heterozygous reached 27.78% (15/54) OsARF24-SG2 site. Phytoene desaturase (PDS) key enzyme involved carotenoid biosynthesis, possessing crucial single-domain (amino acids 106–556). #6 underwent homozygous via OsPDS-SG2, alteration Leucine amino acid 114 Valine, albino phenotype white stripes leaves S3). further explored potential application modulating expression through alternative splicing (AS). As pre-mRNA transcripts undergo processing, AS lead intron retention (IR), 5′ splicing, 3′ exon skipping, offering patterns (Liu both donor (SD) site complementary strand acceptor (SA) harbour T. illustrate application, designed sgRNAs specifically SD SA OsARF24 S4). identified mutant #45 desired within splice 1, which targeted by OsARF24-SG1. performed RT-PCR using primer 1 reverse 3. 240 bp fragment generated wild-type (WT) whereas 319 amplified 1f,g). Sequencing revealed retained, completely prevented production normal isoform 1h). Additionally, 12 mutants 7 will produce lines T1 identifying isoforms Overall, our demonstrate program mutating mature transcripts. explore fusing UNG2Δ88-K184A/N213D/A214V (mhUNGv2) evaluating eight three sequencing 255 caused highly efficient 26.09% 61.11%, including C-to-G 58.33% well C-to-T 40.91%, A, examined S2a–c). percentage C-to-G/T almost exceeded 85%, there very few C-to-A detected S2d). Hence, pCKBE. pCKBE C2-C7, C9-C11, C13 C15-C16 1e), InDel 13.04% 72.22% 8.51%), 22.58%), 50.00%) chimeric 22.58%) 1c; Table evaluate specificity lines, selected off-target based predictions Cas-OFFinder (http://www.rgenome.net/cas-offinder/) targets. Minimal effects observed. Only one OsNRT1.1B-SG3-OFF1 OsLCY-SG3-OFF1 detectable S5). novel excised an producing lines. new C-to-K transversion events. greatly broadened scope breaking narrow window, increasing opportunity obtain strategy research. By utilizing edit (AS) providing approach patterns. InDels compared well-developed pABEs pCBEs. pCBEs facilitate repair following deamination reaction. pCKBE, AYBE CGBE, enabled after generation AP are likely double-stranded breaks during these studies suicide HMCES could reduce byproducts shielding safeguarding CGBE TSBE (He 2024; Huang addition, introducing Gam proteins, bind ends DSBs prevent their degradation, reduced (Komor summary, diverse base-editing toolbox. combining other previously reported editors, types be achieved especially future 1i). This study supported National Natural Science Foundation China (32188102 J.-K.Z.) CAAS Nanfan Research Institute, Chinese Academy Agricultural Sciences (YBXM2424 M.L.). authors declare competing interests. M.L. J.-K.Z. research; Y.W., X.W., H.W., Y.H. Y.W. experiments; C.Z., X.W. transformation; analysed data; wrote manuscript. data supports findings available supplementary material article. Figures S1-S5 Supplementary figures. Tables tables. Please note: publisher responsible content functionality any supporting information supplied authors. Any queries (other missing content) should directed corresponding author

Language: Английский

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Emerging applications of gene editing technologies for the development of climate-resilient crops

Frontiers in Genome Editing, Journal Year: 2025, Volume and Issue: 7

Published: March 10, 2025

Climate change threatens global crop yield and food security due to rising temperatures, erratic rainfall, increased abiotic stresses like drought, heat, salinity. Gene editing technologies, including CRISPR/Cas9, base editors, prime offer precise tools for enhancing resilience. This review explores the mechanisms of these technologies their applications in developing climate-resilient crops address future challenges. While CRISPR/enables targeted modifications plant DNA, editors allow direct conversion without inducing double-stranded breaks, enable insertions, deletions, substitutions. By understanding manipulating key regulator genes involved stress responses, such as DREB, HSP, SOS, ERECTA, HsfA1, NHX; tolerance can be enhanced against salt stress. improve traits related root development, water use efficiency, response pathways, heat shock response, photosynthesis, membrane stability, ion homeostasis, osmotic adjustment, oxidative response. Advancements gene integration with genomics, phenomics, artificial intelligence (AI)/machine learning (ML) hold great promise. However, challenges off-target effects, delivery methods, regulatory barriers must addressed. highlights potential develop crops, contributing sustainable agriculture.

Language: Английский

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Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Plants, Journal Year: 2023, Volume and Issue: 12(7), P. 1478 - 1478

Published: March 28, 2023

Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation transgene-free genome-edited plants in a short period advantages simplicity, high efficiency, specificity, low production costs, which greatly facilitate study gene functions. In plant molecular breeding, efficiency system proven to be key step influencing effectiveness with improvements recently becoming focus reported scientific research. This review details strategies methods improving editing including Cas9 variant enzyme engineering, effect multiple promoter driven Cas9, gRNA efficient optimization expression strategies. It also briefly introduces CRISPR/Cas12a BE PE precision editing. These are beneficial further development systems field breeding.

Language: Английский

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Promoter editing for the genetic improvement of crops

Journal of Experimental Botany, Journal Year: 2023, Volume and Issue: 74(15), P. 4349 - 4366

Published: May 19, 2023

Abstract Gene expression plays a fundamental role in the regulation of agronomically important traits crop plants. The genetic manipulation plant promoters through genome editing has emerged as an effective strategy to create favorable crops by altering pattern pertinent genes. Promoter can be applied directed manner, where nucleotide sequences associated with are precisely generated. Alternatively, promoter also exploited random mutagenic approach generate novel variations within designated promoter, from which elite alleles selected based on their phenotypic effects. Pioneering studies have demonstrated potential engineering well mining valuable for breeding. In this review, we provide update application increased yield, enhanced tolerance biotic and abiotic stresses, improved quality. We discuss several remaining technical bottlenecks how may better employed improvement future.

Language: Английский

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Prime editing: Mechanism insight and recent applications in plants

Plant Biotechnology Journal, Journal Year: 2023, Volume and Issue: 22(1), P. 19 - 36

Published: Oct. 4, 2023

Summary Prime editing (PE) technology utilizes an extended prime guide RNA (pegRNA) to direct a fusion peptide consisting of nCas9 (H840) and reverse transcriptase (RT) specific location in the genome. This enables installation base changes at targeted site using portion pegRNA through RT activity. The resulting product reaction forms 3′ flap, which can be incorporated into genomic series biochemical steps involving DNA repair synthesis pathways. PE has demonstrated its effectiveness achieving almost all precise gene editing, such as conversions (all types), sequence insertions deletions, chromosomal translocation inversion long insertion safe harbour sites within In plant science, could serve groundbreaking tool for allowing creation desired alleles improve crop varieties. Nevertheless, application encountered limitations due efficiency constraints, particularly dicotyledonous plants. this review, we discuss step‐by‐step mechanism PE, shedding light on critical aspects each step while suggesting possible solutions enhance efficiency. Additionally, present overview recent advancements future perspectives research specifically focused plants, examining key technical considerations applications.

Language: Английский

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Recent Advances in Tomato Gene Editing

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(5), P. 2606 - 2606

Published: Feb. 23, 2024

The use of gene-editing tools, such as zinc finger nucleases, TALEN, and CRISPR/Cas, allows for the modification physiological, morphological, other characteristics in a wide range crops to mitigate negative effects stress caused by anthropogenic climate change or biotic stresses. Importantly, these tools have potential improve crop resilience increase yields response challenging environmental conditions. This review provides an overview techniques used plants, focusing on cultivated tomatoes. Several dozen genes that been successfully edited with CRISPR/Cas system were selected inclusion illustrate possibilities this technology improving fruit yield quality, tolerance pathogens, responses drought soil salinity, among factors. Examples are also given how domestication wild species can be accelerated using generate new better adapted climatic situation suited indoor agriculture.

Language: Английский

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CRISPR/CasΦ2‐mediated gene editing in wheat and rye

Journal of Integrative Plant Biology, Journal Year: 2024, Volume and Issue: 66(4), P. 638 - 641

Published: Feb. 13, 2024

The recent advancements in developing the CRISPR/Cas9 system and various derivative tools (e.g., base editors) have accelerated basic plant science research crop improvement by creating multiple types of genetic variations (Li et al., 2023a). However, use Cas9 protein is frequently limited requirement G/C-rich protospacer-adjacent motif (PAM) sequences, especially triticeae plants, many which are important food forage crops carrying large complex genomes. CRISPR/CasΦ (CRISPR/Cas12j) has recently been discovered from bacteriophages, prefers 5′-TBN-3′ PAMs suitable for specific biological therapeutic applications (Pausch 2020). With a smaller size (700–800 aa) than Cas12a (1,100 SpCas9 (1,300 aa), it valuable DNA editing where or nucleic acid limiting factor (Zhan 2021). Lately, CRISPR/CasΦ2 demonstrated useful gene transient transgenic experiments Arabidopsis, tomato, rice, maize (Liu 2022; Li 2023b). remains unclear whether may function plants. More importantly, worth exploring if this hypercompact be adopted precise plants (Figure 1A). Improving (A) CRISPR/CasΦ2-meadited toolkits. (B) Comparison frequency among pBlunt-CasΦ2Ta-V1/V2/V3/V4 at TaGW2 target sites (TBN PAMs) with TaU3-tRNA-crRNA wheat protoplasts. Representative deletion locations were summarized using TaGW2-crRNA-TTN/pBlunt-CasΦ2Ta-V3 data. (C) Assessment frequencies CRISPR/CasΦ2Ta-V3 CRISPR/VCasΦ2Ta-V3 TaGW2-crRNA-TTN site (D) Test TaGW2/ScPhyA-crRNA-TTN paired opposite crRNAs rye (E) Diagram CasΦ2/dCasΦ2 VCasΦ2/dVCasΦ2-based CBEs. (F) Frequencies C-to-T indels obtained four CBEs (G) frequencies, window five different pBlunt-dCasΦ2Ta-CBE (H) dCasΦ2-based adenine editor (ABE). (I) A-to-G inducing ABE (J) CasΦ2/dCasΦ2-derived cytosine editors (CBEs) TaGW2/TaPIN-crRNA-TTN dCasΦ2-derived TaALS-crRNA-TTN All values mean ± s.e.m. *P < 0.05, **P 0.01; ns, no significant difference two-tailed Student's t test. To address above questions, we first synthesized codon-optimized CasΦ2 constructed UBQ::CasΦ2Ta TaU3::crRNA cassette placed into pBlunt vector S1A). We chose genes editing: two (TaGW2 TaPIN) (ScPhyA ScPhyB). As B G, T C, designed three each TTN, TGN, TCN PAM (creating 12 sites), covering similar crRNA binding sites. When testing protoplasts-based systems, obvious was detected PCR/RE assays any S1B). This line very low efficiency (<1%) genome Arabidopsis Consequently, set out to improve CRISPR/CasΦ2. First, changed way processing tested nuclear localization signals (NLSs) CasΦ2. By combining TaU3 promoter-driven polycistronic-tRNA-crRNA NLSs (SV40 nucleoplasmin long NLS) fused CasΦ2Ta, generated versions CRISPR/CasΦ2Ta 1B, V2, V3, V4). protoplasts sites, found that pBlunt-TaU3-tRNA-crRNA/pBlunt-CasΦ2Ta-V3, one N- C-termini respectively, could most efficiently near-background level up 3.2% 1B). Considering CasΦ variants NCasΦ VCasΦ cleave substrate faster 2021), next prepared new constructs expressing NCasΦ2Ta-V3 VCasΦ2Ta-V3 their efficiencies ScPhyA protoplasts, appropriate pBlunt-TaU3-tRNA-crRNA S2A). showed both improved compared CasΦTa-V3, exhibiting an overall higher (2.5–6.0-fold increase, Figure S2B, C). Hence T-DNA constructs, pLH-CasΦ2Ta-V3 pLH-VCasΦ2Ta-V3, TaU3::tRNA-crRNA design S3A). Sanger sequencing analysis indicated pLH-VCasΦ2Ta-V3 induced 30% (6/20) T0 did (12.5%, 2/16) 1C), mutations being 3–27 bp deletions S3B). Analysis T1 verified inheritance indel pLH-VCasΦ2Ta-V3-TaGW2-crRNA-TTN generation (Table S3). Interestingly, observed crRNAs, arranged as 5′-TTN-N18-spacing-N18-YAA-3′, further pBlunt-VCasΦ2Ta-V3 assays. ScPhyA-crRNA-TTN, ~30 ~60-bp spacing sequences 1D). (~30 sequence) ~1.5-fold produced single Altogether, results suggest enables rye, potential crRNAs. Furthermore, endeavored develop (CBE ABE) CasΦ2Ta-V3. created catalytically inactive dCasΦ2Ta-V3 mutating active RuvC domain (D394, E606, D695) Then human APOBEC3A CasΦ2Ta-V3 (Zong 2018), pBlunt-CasΦ2Ta-CBE 1E). investigate VCasΦ2Ta influence CBE, also pBlunt-VCasΦ2Ta-CBE pBlunt-dVCasΦ2Ta-CBE vectors, dVCasΦ2Ta-V3 developed similarly dCasΦ2Ta-V3. site. Deep (up ~4%) levels 1F). expected, exhibited high Thus, another employed test In these assays, CBE ranged 1.9% 5.5%, spanning C2 C17 protospacers 1G), wider reported Cas9-based 2018). Therefore, pLH-dCasΦ2Ta-CBE pLH-CasΦ2Ta-CBE S5A), TaPIN-crRNA-TTN pLH-dCasΦ2Ta-CBE, activities 9.1% 6.9% TaPIN-crRNA-TTN, only C substitution 1J). contrast, mutants pLH-CasΦ2Ta-CBE-TaGW2-crRNA-TTN, 80% mostly transmitted For evaluating usefulness dCasΦ2-ABE, TadA8e XTEN linker (Yan thus generating pBlunt-dCasΦ2Ta-ABE 1H). investigated all targets (in TaALS, TaNAC2, ScPhyB, ScPhyC, respectively), ranging 0.8% 3.0% 1I). deamination spanned protospacer positions A9–A11, unwanted Hence, pLH-dCasΦ2Ta-ABE S6A) examine activity 6% (Figures 1J, S6B), Collectively, illustrated feasibility rye. Finally, examined off-targeting CRISPR/VCasΦ2Ta-V3, CRISPR/dCasΦ2Ta-CBE CRISPR/dCasΦ2Ta-ABE mediated mutants. used analysis, eight (1–4 mismatches; Table S4) identified common genome. revealed off-target events S7A, B), indicating specificity wheat. summary, changes expression, NLS incorporation, variants, resulting successful knockout specificity. proved time CRISPR/dCasΦ2-CBE CRISPR/dCasΦ2-ABE functional its unique properties, i.e., efficient TTN alternative window, provides complementary engineering tool, find wide future on CRISPR/CasΦ2-mediated modifications. work supported National Key Research Development Program China (2021YFF1000203) Natural Science Foundation (32000286 32370432). authors declare conflict interest. S.Z., X.H., Y.Z., Y.H., H.Liu., Z.C., H.Li., Dan.W., C.T., Y.Y., Y.G. performed experiments. X.J., Dao.W., X.S. conceived project wrote manuscript. approved final Additional Supporting Information online supporting information tab article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13624/suppinfo S1. pBlunt-TaU3-crRNA pBlunt-CasΦ2Ta-V1 vectors S2. pBlunt-CasΦ2Ta-V3, pBlunt-NCasΦ2Ta-V3 assessment S3. representative genotypes S4. strategy increasing pBlunt-VCasΦ2Ta-V3-mediated assay S5. diagram S6. S7. CRISPR/VCasΦ2Ta-V3-TaGW2-crRNA-TTN, CRISPR/dCasΦ2Ta-CBE-TaGW2-crRNA-TTN CRISPR/dCasΦ2Ta-ABE-TaALS-crRNA-TTN indel, primer constructing study primers preparing mutation transmission derived pLH-CasΦ2Ta-CBE, progenies Potential barcodes deep Other Please note: publisher not responsible content functionality supplied authors. Any queries (other missing content) should directed corresponding author article.

Language: Английский

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