High performance TadA-8e derived cytosine and dual base editors with undetectable off-target effects in plants

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

Published: June 14, 2024

Abstract Cytosine base editors (CBEs) and adenine (ABEs) enable precise C-to-T A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances edits in mammalian cells plants. Interestingly, TadA-8e can also be evolved to confer editing. This study compares engineered CBEs rice tomato cells, identifying TadCBEa, TadCBEd, TadCBEd_V106W as efficient with high purity a narrow editing window. A dual editor, TadDE, promotes simultaneous Multiplexed TadCBEa TadDE is demonstrated transgenic rice, no off-target effects detected by whole genome transcriptome sequencing, indicating specificity. Finally, two crop engineering applications using are shown: introducing herbicide resistance alleles OsALS creating synonymous mutations OsSPL14 resist OsMIR156 -mediated degradation. Together, this presents editor valuable additions the plant toolbox.

Language: Английский

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Expanding plant genome editing scope and profiles with CRISPR‐FrCas9 systems targeting palindromic TA sites

Plant Biotechnology Journal, Journal Year: 2024, Volume and Issue: 22(9), P. 2488 - 2503

Published: May 7, 2024

Summary CRISPR‐Cas9 is widely used for genome editing, but its PAM sequence requirements limit efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) plant especially in rice. FrCas9 recognizes a concise 5′‐NNTA‐3′ PAM, targeting more abundant palindromic TA sites genomes than the 5′‐NGG‐3′ of most popular SpCas9. shows cleavage activities at all tested with editing outcomes sharing same characteristics typical system. induces high‐efficiency targeted mutagenesis stable rice lines, readily generating biallelic mutants expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion exonuclease, TREX2. TREX2‐FrCas9 generates much without compromise demonstrate as an efficient tool genetic knockout microRNA gene. Furthermore, FrCas9‐derived cytosine base editors (CBEs) and adenine (ABE) are developed produce C‐to‐T A‐to‐G edits plants. Whole‐genome sequencing‐based off‐target analysis suggests that highly specific nuclease. Expression plants, however, causes detectable guide RNA‐independent mutations, mostly single nucleotide variants (SNVs). Together, have established CRISPR‐FrCas9 system mutagenesis, large deletions, The simple motif makes promising plants expanded scope.

Language: Английский

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Crop Improvement: Comparison of Transgenesis and Gene Editing

Horticulturae, Journal Year: 2024, Volume and Issue: 10(1), P. 57 - 57

Published: Jan. 6, 2024

The development and improvement of molecular biology methods have led to the creation new technologies that make it possible modify plant genomes by transferring integrating into genomes’ heterologous genes from various expression systems (genetic engineering), as well inducing knockouts one or more target interest (genomic editing). genome-editing is a milestone in modern breeding certainly relies on knowledge developed for transgenesis. This review will discuss issues related advantages disadvantages both improving economically valuable traits important crops.

Language: Английский

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The elite haplotype OsGATA8-H coordinates nitrogen uptake and productive tiller formation in rice

Nature Genetics, Journal Year: 2024, Volume and Issue: 56(7), P. 1516 - 1526

Published: June 13, 2024

Abstract Excessive nitrogen promotes the formation of nonproductive tillers in rice, which decreases use efficiency (NUE). Developing high-NUE rice cultivars through balancing uptake and productive remains a long-standing challenge, yet how these two processes are coordinated elusive. Here we identify transcription factor OsGATA8 as key coordinator tiller rice. negatively regulates by repressing ammonium transporter gene OsAMT3.2 . Meanwhile, it OsTCP19 , negative modulator tillering. We -H haplotype with enhanced higher proportion tillers. The geographical distribution OsGATA8- H its frequency change historical accessions suggest adaption to fertile soil. Overall, this study provides molecular evolutionary insights into regulation NUE facilitates breeding NUE.

Language: Английский

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Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Sept. 29, 2023

Abstract Among CRISPR-Cas genome editing systems, Streptococcus pyogenes Cas9 (SpCas9), sourced from a human pathogen, is the most widely used. Here, through in silico data mining, we have established an efficient plant engineering system using CRISPR-Cas9 probiotic Lactobacillus rhamnosus . We confirmed predicted 5’-NGAAA-3’ PAM via bacterial depletion assay and showcased its exceptional efficiency rice, wheat, tomato, Larix cells, surpassing LbCas12a, SpCas9-NG, SpRY when targeting identical sequences. In stable rice lines, LrCas9 facilitates multiplexed gene knockout coding sequence achieves knockdown targeted promoter deletion, demonstrating high specificity. also developed LrCas9-derived cytosine adenine base editors, expanding capabilities. Finally, by harnessing LrCas9’s A/T-rich preference, created CRISPR interference activation systems plants. Together, our work establishes CRISPR-LrCas9 as user-friendly tool for diverse applications crops beyond.

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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Efficient and multiplex gene upregulation in plants through CRISPR-Cas-mediated knockin of enhancers

Molecular Plant, Journal Year: 2024, Volume and Issue: 17(9), P. 1472 - 1483

Published: July 24, 2024

Language: Английский

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IsDge10 is a hypercompact TnpB nuclease that confers efficient genome editing in rice

Plant Communications, Journal Year: 2024, Volume and Issue: 5(11), P. 101068 - 101068

Published: Aug. 23, 2024

Language: Английский

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Dissection of the IDA promoter identifies WRKY transcription factors as abscission regulators in Arabidopsis

Journal of Experimental Botany, Journal Year: 2024, Volume and Issue: 75(8), P. 2417 - 2434

Published: Jan. 31, 2024

Abstract Plants shed organs such as leaves, petals, or fruits through the process of abscission. Monitoring cues age, resource availability, and biotic abiotic stresses allow plants to abscise in a timely manner. How these signals are integrated into molecular pathways that drive abscission is largely unknown. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene one main drivers floral organ Arabidopsis known transcriptionally respond most abscission-regulating cues. By interrogating IDA promoter silico vitro, we identified transcription factors could potentially modulate expression. We probed importance ERF- WRKY-binding sites for expression during abscission, with WRKYs being special relevance mediate up-regulation response stress tissues destined separation. further characterized WRKY57 positive regulator IDA-like zones. Our findings highlight promise element-targeted approaches responsiveness signaling pathway harness controlled timing improved crop productivity.

Language: Английский

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Boosting genome editing in plants with single transcript unit surrogate reporter systems

Plant Communications, Journal Year: 2024, Volume and Issue: 5(6), P. 100921 - 100921

Published: April 15, 2024

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low activity complicates identification events. In this study, we introduce multiple single transcript unit surrogate reporter (STU-SR) systems to enhance selection genome-edited plants. These use same guide RNAs designed endogenous genes edit genes, establishing a direct link between gene that genes. Various strategies are used restore functional after editing, including efficient single-strand annealing (SSA) homologous recombination in STU-SR-SSA systems. STU-SR-base editor leverage base reinstate start codon, enriching C-to-T A-to-G Our results showcase effectiveness these STU-SR enhancing events monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine adenine editing. The exhibit compatibility with variants, such as PAM-less SpRY, shown boost Brassica oleracea, dicot vegetable crop. summary, have developed highly versatile enrichment

Language: Английский

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