Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance

Journal of Integrative Plant Biology, Journal Year: 2023, Volume and Issue: 65(9), P. 2194 - 2203

Published: July 4, 2023

Abstract Exploiting novel endogenous glyphosate‐tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here, through fusions of different effective cytosine adenine deaminases with nCas9‐NG, we engineered an surrogate two‐component composite base editing system, STCBE‐2, improved C‐to‐T A‐to‐G efficiency expanded the window. Furthermore, targeted a OsEPSPS gene artificial evolution STCBE‐2‐mediated near‐saturated mutagenesis. After hygromycin glyphosate selection, identified allele Asp‐213‐Asn (D213N) mutation ( ‐D213N) predicted glyphosate‐binding domain, which conferred plants reliable tolerance had not been reported or applied Collectively, developed dual editor will be valuable important genes crops. And germplasm generated this study benefit weeds management paddy fields.

Language: Английский

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Engineering a plant A-to-K base editor with improved performance by fusion with a transactivation module

Plant Communications, Journal Year: 2023, Volume and Issue: 4(6), P. 100667 - 100667

Published: July 31, 2023

Language: Английский

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Advances in cis-element- and natural variation-mediated transcriptional regulation and applications in gene editing of major crops

Journal of Experimental Botany, Journal Year: 2023, Volume and Issue: 74(18), P. 5441 - 5457

Published: June 29, 2023

Transcriptional regulation is crucial to control of gene expression. Both spatio-temporal expression patterns and levels genes are determined by the interaction between cis-acting elements trans-acting factors. Numerous studies have focused on factors that mediate transcriptional regulatory networks. However, elements, such as enhancers, silencers, transposons, natural variations in genome, also vital for could be utilized clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing improve crop quality yield. In this review, we discuss current understanding cis-element-mediated major crops, including rice (Oryza sativa), wheat (Triticum aestivum), maize (Zea mays), well latest advancements techniques their applications crops highlight prospective strategies breeding.

Language: Английский

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Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Aug. 23, 2024

Type I CRISPR-Cas systems are widespread and have exhibited high versatility efficiency in genome editing gene regulation prokaryotes. However, due to the multi-subunit composition large size, their application eukaryotes has not been thoroughly investigated. Here, we demonstrate that type I-F2 Cascade, most compact among systems, with a total size smaller than of SpCas9, can be developed for transcriptional activation human cells. The engineered tool match or surpass dCas9. Additionally, create base editor using which induces considerably wide window (~30 nt) bimodal distribution. It expand targetable sites, is useful disrupting functional sequences genetic screening. This research underscores eukaryotes, particularly development window. widely distributed prokaryotes but long overlooked applications eukaryotes. authors develop activators editors windows cells Cascades

Language: Английский

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Efficient and heritable A-to-K base editing in rice and tomato

Horticulture Research, Journal Year: 2023, Volume and Issue: 11(1)

Published: Dec. 11, 2023

Abstract Cytosine and adenosine base editors (CBE ABE) have been widely used in plants, greatly accelerating gene function research crop breeding. Current can achieve efficient A-to-G C-to-T/G/A editing. However, heritable A-to-Y (A-to-T/C) editing remains to be developed plants. In this study, a series of A-to-K editor (AKBE) systems were constructed for monocot dicot Furthermore, nSpCas9 was replaced with the PAM-less Cas9 variant (nSpRY) expand target range AKBEs. Analysis 228 T0 rice plants 121 tomato edited using AKBEs at 18 endogenous loci revealed that, addition highly substitution (41.0% on average), plant A-to-T conversion efficiencies up 25.9 10.5% tomato, respectively. Moreover, rice-optimized AKBE generates A-to-C rice, an average efficiency 1.8%, revealing significant value plant-optimized creating genetic diversity. Although most edits chimeric, desired types could transmitted T1 offspring, similar generated by traditional ABE8e. Besides, tyrosine (Y, TAT) or cysteine (C, TGT) achieved introduction early stop codon (TAG/TAA/TGA) genes, demonstrating its potential use disruption.

Language: Английский

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Genome Editing: A Novel Breeding Strategy for Enhanced Stress Tolerance and Quality in Oilseed Crops

Published: Jan. 1, 2025

Language: Английский

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Engineering a bacterial toxin deaminase from the DYW-family into a novel cytosine base editor for plants and mammalian cells

Genome biology, Journal Year: 2025, Volume and Issue: 26(1)

Published: Feb. 3, 2025

Abstract Base editors are precise editing tools that employ deaminases to modify target DNA bases. The DYW-family of cytosine is structurally and phylogenetically distinct might be harnessed for genome tools. We report a novel CRISPR/Cas9-cytosine base editor using SsdA, DYW-like deaminase bacterial toxin. A G103S mutation in SsdA enhances C-to-T efficiency while reducing its toxicity. Truncations result an extraordinarily small enzyme. SsdA-base efficiently converts rice barley protoplasts induces mutations plants mammalian cells. engineered highly efficient tool.

Language: Английский

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Multiplex genome editing for climate-resilient woody plants

Frontiers in Forests and Global Change, Journal Year: 2025, Volume and Issue: 8

Published: Feb. 5, 2025

Climate change is severely impacting global forest ecosystems, stressing woody plants due to rising temperatures, shifting precipitation patterns, and extreme weather events. These pressures threaten biodiversity disrupt the essential roles forests play in carbon sequestration, timber production, ecosystem stability. Traditional management strategies, such as selective breeding, cannot keep up with rapid pace of climate change, given long juvenile phase trees. Multiplex genome editing, particularly through CRISPR technologies, offers a promising solution accelerate development climate-resilient traits plants. By simultaneously targeting multiple genes, multiplex enables efficient modification polygenic that govern stress tolerance, disease resistance, other crucial resilience factors. This mini-review examines potential technologies management, agroecological practices, showing how they can improve tree support sustainable forestry response growing challenges change.

Language: Английский

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An optimized thymine base editing toolkit with various editing windows enables targeted T‐to‐G base conversions in rice

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

Published: Feb. 17, 2025

Base editors offer potential for site saturation mutagenesis, yet deaminase-based are constrained to adenine and cytosine targets (Li et al., 2023a). Recently, glycosylase-based base (gBEs), which fuse engineered glycosylases with SpCas9 nickase (SpCas9n, D10A) excise specific guanine or thymine bases, achieve conversions through DNA repair over abasic sites (He 2024; Tong 2023; Ye 2024). While (gGBEs) show efficient conversion in plants (Liu Tian 2024), (TBEs) remain unexplored (Figure S1). Previous studies identified that the Y147A mutation human uracil glycosylase (hUNG) produces a variant (hTDG). Highly active variants, TDG-EK 2024) TDG3 (Ye were using protein-language-assisted design directed evolution, respectively, enhance editing. Cas9-embedding strategy further enhances editing efficiency mammalian cells 1a; He To engineer TBE tools plants, we inserted three plant-codon-optimized TDG variants (hTDG, TDG3; Figure 1b) into SpCas9n at various positions (I1029-G1030, F1046-I1063 P1249-E1250) GGGGS linker. Initially, assessed of TDG3) rice protoplasts dual-luciferase reporter system, hUNG as control. The system utilized ZmUBI-driven firefly luciferase (FLuc) reference 2x35S-driven NanoLuc (NLuc) reporter. A nonsense was introduced Gly69 (GGA > TGA), converted read-through codon by successful editing, restoring NLuc translation S2). Three sgRNAs designed edit (T9, T10 T11; 1c). Transient assays revealed outperformed hTDG 2.2- 20.8-fold 1.3- 4.5-fold, CE1046TDG3 showing highest activity T9 1d, S3). evaluate efficiencies Cas9-embbeded TBEs (CE-TBEs) transgenic tested six endogenous (SLR1-g1, SLR1-g2, SLR1-g3, ALS1-g1, EPSPS-g1, TB1-g1; Table total 1681 T0 edited nine CE-TBEs regenerated analysed via Hi-TOM sequencing (Table Plants chimerism >10% considered valid edits S4) confirmed subcloning S5). Genotyping induced only 3.8% 1.5% indels SLR1-g2 respectively 1e). For variant, CE1029TDG-EK CE1249TDG-EK constructs showed no detectable activity, while CE1046TDG-EK achieved T-to-G transversion T11 SLR1-g3 T3 TB1-g1 3.6% (1/28) 2.1% (1/48), 1f). Consistent protoplast results, significantly enhanced rice, increasing indel 1e,f). CE1029TDG3 CE1249TDG3, average 29.1% 16.4% 1g). 38.2% (13/34) CE-TDG3 predominantly yielded except CE1046TDG3, 5.9% (2/34) T-to-A target. No T-to-C detected. Notably, different embedding exhibited distinct windows. wide editable range (T-6 T17; PAM located 21–23; 1h), displayed narrower window (T-1 1i). CE1249TDG3 led backward-shifted (mainly T9–T14; 1j). At SLR3-g3 target, high proportion amino acid substitutions in-frame deletions TVHYNP motif exhibit semi-dwarf phenotype 1k,l, S6). observed within protospacers all where CE1029TDG3, proportions mutations outside protospacer on targeted strand 1m,n, Although most chimeric, likely due sustained T-DNA-free mutants obtained T1 generation 1o, S4). In this study, compared series Cas9-embedded developed TBEs: CE1249TDG3. demonstrated enabled windows, achieving up rice. Unlike (predominantly T-to-C/G; Escherichia coli T-to-A; our rice-optimized (CE-TDG3) primarily significant off-target Improving would purity (Tong Combining ABE AKBE 2023b; Tan 2022; Wu 2023) allows T:A base-pair any desired 1p), broadening possibilities generating elite germplasm. This study supported Hainan Seed Industry Laboratory (B22C1000P Y.T.) Yazhouwan National (2410KX01 K.T.). Y.T. X.L. research; X.L., R.S. performed experiments; X.L data; Y.T., Y.P. K.T. wrote revised manuscript. authors declare competing interests. plasmids (CE1029hTDG, CE1029TDG-EK, CE1046hTDG, CE1046TDG-EK, CE1249hTDG, CE1249TDG-EK, NTDG3 CTDG3) constructed available upon request corresponding author ([email protected]). NGS data deposited Genomics Data Center (NGDC; https://ngdc.cncb.ac.cn/) under accession number PRJCA030169 PRJCA034519. S1 Composition current editors. S2 Identification results NLuc. S3 Comparison fusion positions. S4. Representative amplicon selected plants. S5 S6 Sequence sequences related vectors genes. sequence cassette pDLuc-TBE. Summary sites. HiTOM S4 study. Heritability analysis progenies. Primers oligos used Please note: publisher is not responsible content functionality supporting information supplied authors. Any queries (other than missing content) should be article.

Language: Английский

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Current Advancement and Future Prospects in Simplified Transformation-Based Plant Genome Editing

Plants, Journal Year: 2025, Volume and Issue: 14(6), P. 889 - 889

Published: March 12, 2025

Recent years have witnessed remarkable progress in plant biology, driven largely by the rapid evolution of CRISPR/Cas-based genome editing (GE) technologies. These tools, including versatile CRISPR/Cas systems and their derivatives, such as base editors prime editors, significantly enhanced universality, efficiency, convenience functional genomics, genetics, molecular breeding. However, traditional genetic transformation methods are essential for obtaining GE plants. depend on tissue culture procedures, which time-consuming, labor-intensive, genotype-dependent, challenging to regenerate. Here, we systematically outline current advancements simplifying GE, focusing optimization process through developmental regulators, development planta methods, establishment nanomaterial- viral vector-based delivery platforms. We also discuss critical challenges future directions achieving genotype-independent, culture-free GE.

Language: Английский

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