PAM‐relaxed and temperature‐tolerant CRISPR‐Mb3Cas12a single transcript unit systems for efficient singular and multiplexed genome editing in rice, maize, and tomato DOI Creative Commons
Shishi Liu, Yao He, Tingting Fan

et al.

Plant Biotechnology Journal, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 10, 2024

Class 2 Type V-A CRISPR-Cas (Cas12a) nucleases are powerful genome editing tools, particularly effective in A/T-rich genomic regions, complementing the widely used CRISPR-Cas9 plants. To enhance utility of Cas12a, we investigate three Cas12a orthologs-Mb3Cas12a, PrCas12a, and HkCas12a-in Protospacer adjacent motif (PAM) requirements, efficiencies, profiles compared rice. Among these orthologs, Mb3Cas12a exhibits high efficiency at target sites with a simpler, relaxed TTV PAM which is less restrictive than canonical TTTV LbCas12a AsCas12a. optimize Mb3Cas12a, develop an efficient single transcription unit (STU) system by refining linker between CRISPR RNA (crRNA), nuclear localization signal (NLS), direct repeat (DR). This optimized enables precise rice, for fine-tuning gene expression promoter regions. Further, introduced Arginine (R) substitutions Aspartic acid (D) 172, Asparagine (N) 573, Lysine (K) 579 creating two temperature-tolerant variants: Mb3Cas12a-R (D172R) Mb3Cas12a-RRR (D172R/N573R/K579R). These variants demonstrate significantly improved lower temperatures (22 °C 28 °C) rice cells, showing best performance. We extend this approach developing STU systems maize tomato, achieving biallelic mutants targeting or multiple genes T

Language: Английский

Recent advances of CRISPR-based genome editing for enhancing staple crops DOI Creative Commons
Feng Chen, Chen D. Lu, Yan Zhao

et al.

Frontiers in Plant Science, Journal Year: 2024, Volume and Issue: 15

Published: Sept. 23, 2024

An increasing population, climate change, and diminishing natural resources present severe threats to global food security, with traditional breeding genetic engineering methods often falling short in addressing these rapidly evolving challenges. CRISPR/Cas systems have emerged as revolutionary tools for precise modifications crops, offering significant advancements resilience, yield, nutritional value, particularly staple crops like rice maize. This review highlights the transformative potential of technology, emphasizing recent innovations such prime base editing, development novel CRISPR-associated proteins, which significantly improved specificity, efficiency, scope genome editing agriculture. These enable targeted that enhance tolerance abiotic stresses well biotic stresses. Additionally, plays a crucial role improving crop yield quality by enhancing photosynthetic nutrient uptake, resistance lodging, while also taste, texture, shelf life, content through biofortification. Despite challenges off-target effects, need more efficient delivery methods, ethical regulatory concerns, underscores importance security sustainability It calls continued research integration CRISPR other emerging technologies nanotechnology, synthetic biology, machine learning fully realize its developing resilient, productive, sustainable agricultural systems.

Language: Английский

Citations

10
CRISPR–Cas applications in agriculture and plant research DOI
Aytug Tuncel, Changtian Pan,

Joshua S. Clem

et al.

Nature Reviews Molecular Cell Biology, Journal Year: 2025, Volume and Issue: unknown

Published: March 7, 2025

Language: Английский

Citations

1
Exploiting the efficient Exo:Cas12i3‐5M fusions for robust single and multiplex gene editing in rice DOI Creative Commons

Wenxue Wang,

Shaoya Li,

Jiaying Yang

et al.

Journal of Integrative Plant Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 28, 2025

ABSTRACT The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement. CRISPR/Cas12i3, which belongs to the Class II Type V‐I Cas system, has attracted extensive attention recently due its smaller protein size less restricted canonical “TTN” protospacer adjacent motif (PAM). However, relatively lower efficiency, Cas12i3‐mediated not yet been documented plants. Here, we fused four 5′ exonucleases (Exo) including T5E, UL12, PapE, ME15 N terminal an optimized Cas12i3 variant (Cas12i3‐5M), respectively, systematically evaluated activities these Exo:Cas12i3‐5M fusions across six endogenous targets rice stable lines. We demonstrated that increased efficiencies by up 12.46‐fold 1.25‐fold compared with Cas12i3‐5M, respectively. Notably, UL12:Cas12i3‐5M fusion enabled robust 90.42%–98.61% tested genes. further that, although all Exo:Cas12i5‐5M were capable editing, exhibited superior performance simultaneous three, four, five genes 82.76%, 61.36%, 52.94%, 51.06% lines, Together, different systemically established as more rice. alternative will enrich plant genome toolkits facilitate agronomically important traits

Language: Английский

Citations

0
Systematic high-throughput evaluation reveals FrCas9’s superior specificity and efficiency for therapeutic genome editing DOI
Rui Tian, Xun Tian,

Yang Meiying

et al.

Science Advances, Journal Year: 2025, Volume and Issue: 11(13)

Published: March 26, 2025

CRISPR-Cas9 systems have revolutionized genome editing, but the off-target effects of Cas9 limit its use in clinical applications. Here, we systematically evaluate FrCas9, a variant from Faecalibaculum rodentium , for cell and gene therapy (CGT) applications compare performance to SpCas9 OpenCRISPR-1. OpenCRISPR-1 is CRISPR system synthesized de novo using large language models (LLMs) has not yet undergone systematic characterization. Using AID-seq, Amplicon sequencing, GUIDE-seq, assessed on-target activity profiles these across multiple genomic loci. FrCas9 demonstrated higher efficiency substantially fewer than Furthermore, TREX2 fusion with reduced deletions translocations, enhancing stability. Through screening 1903 sgRNAs targeting 21 CGT-relevant genes sequential GUIDE-seq analysis, identified optimal each gene. Our high-throughput platform highlights particularly TREX2-fused form, as highly specific efficient tool precise therapeutic editing.

Language: Английский

Citations

0
An Agrobacterium‐mediated base editing approach generates transgene‐free edited banana DOI
Senne Van den Broeck, Yvan Ngapout, Bart Panis

et al.

New Phytologist, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Genome editing for the development of improved varieties is supported by possibility segregating out editor T-DNA cassette after genome in many crop species. Removal prevents potential continuous activity transformed plant and furthermore facilitates regulatory approval. While transgene outcrossing exogenous sequences possible crops, this not case vegetatively propagated sterile such as Cavendish bananas. Therefore, gene techniques leading to transgene-free edited plants are essential untap those crops. Here, we present a method banana (Musa spp.) through co-editing strategy. A novel Agrobacterium tumefaciens-mediated approach combining embryogenesis chlorsulfuron selection was established validated whole sequencing. Editing acetolactate synthase (MaALS) genes using base allows effective plants. Moreover, plantlets were regenerated with mutations at two target sites, indicating that strategy can be used multiple genomic sites. The presented efficient represents first report

Language: Английский

Citations

0
Engineering a robust Cas12i3 variant‐mediated wheat genome editing system DOI Creative Commons
Wenxue Wang, Lei Yan, Jingying Li

et al.

Plant Biotechnology Journal, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 17, 2024

Wheat (Triticum aestivum L., 2n = 6x 42, AABBDD) is one of the most important food crops in world. CRISPR/Cas12i3, which belongs to type V-I Cas system, has attracted extensive attention recently due its smaller protein size and less-restricted canonical 'TTN' protospacer adjacent motif (PAM). However, relatively lower editing efficacy plants hexaploidy complex nature wheat, Cas12i3/Cas12i3-5M-mediated genome wheat not been documented yet. Here, we report engineering a robust Cas12i3-5M-mediated system through fusion T5 exonuclease (T5E) combination with an optimised crRNA expression strategy (Opt). We first showed that T5E, rather than ExoI, Cas12i3-5M increased gene efficiencies by up 1.34-fold 3.87-fold, compared Cas12i3 HEK293T cells, respectively. efficiency remains low wheat. then demonstrated Opt-T5E-Cas12i3-5M could enhance 1.20- 1.33-fold 4.05- 7.95-fold stable lines Opt-Cas12i3-5M Opt-Cas12i3, respectively, progressive 5'-end resection DNA strand at cleavage site deletion size. The enabled ranging from 60.71% 90.00% across four endogenous target genes three elite Chinese varieties. Together, developed enriches toolkits for either biological research or genetic improvement may be extended other polyploidy crop species.

Language: Английский

Citations

3
Versatile plant genome engineering using anti-CRISPR-Cas12a systems DOI
Yao He, Shishi Liu, Long Chen

et al.

Science China Life Sciences, Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 15, 2024

Language: Английский

Citations

2
An efficient CRISPR‐Cas12a‐mediated MicroRNA knockout strategy in plants DOI Creative Commons
Xuelian Zheng, Xu Tang, Yuechao Wu

et al.

Plant Biotechnology Journal, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 14, 2024

Summary In recent years, the CRISPR‐Cas9 nuclease has been used to knock out MicroRNA (miRNA) genes in plants, greatly promoting study of miRNA function. However, due its propensity for generating small insertions and deletions, Cas9 is not well‐suited achieving a complete knockout genes. By contrast, CRISPR‐Cas12a generates larger which could significantly disrupt secondary structure pre‐miRNA prevent production mature miRNAs. Through case OsMIR390 rice, we confirmed that Cas12a more efficient tool than mutants gene. To further demonstrate CRISPR‐Cas12a‐mediated targeted nine OsMIRNA have different spaciotemporal expression previously investigated via genetic approaches. With CRISPR‐Cas12a, up 100% genome editing efficiency was observed at these loci. The resulting deletions suggest robustly generated null alleles Transcriptome profiling mutants, as well phenotypic analysis rice grains revealed function miRNAs controlling gene regulating grain quality seed development. This established an plants.

Language: Английский

Citations

2
Advancements of CRISPR-Mediated Base Editing in Crops and Potential Applications in Populus DOI Open Access
Xuefei Yang, Ping Zhu, Jinshan Gui

et al.

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(15), P. 8314 - 8314

Published: July 30, 2024

Base editing represents a cutting-edge genome technique that utilizes the CRISPR system to guide base deaminases with high precision specific genomic sites, facilitating targeted alteration of individual nucleotides. Unlike traditional gene approaches, does not require DNA double-strand breaks or donor templates. It functions independently cellular repair machinery, offering significant advantages in terms both efficiency and accuracy. In this review, we summarize core design principles various editors, their distinctive characteristics, tactics refine efficacy. We also applications crop genetic improvement explore potential contributions forest engineering.

Language: Английский

Citations

1
PAM‐relaxed and temperature‐tolerant CRISPR‐Mb3Cas12a single transcript unit systems for efficient singular and multiplexed genome editing in rice, maize, and tomato DOI Creative Commons
Shishi Liu, Yao He, Tingting Fan

et al.

Plant Biotechnology Journal, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 10, 2024

Class 2 Type V-A CRISPR-Cas (Cas12a) nucleases are powerful genome editing tools, particularly effective in A/T-rich genomic regions, complementing the widely used CRISPR-Cas9 plants. To enhance utility of Cas12a, we investigate three Cas12a orthologs-Mb3Cas12a, PrCas12a, and HkCas12a-in Protospacer adjacent motif (PAM) requirements, efficiencies, profiles compared rice. Among these orthologs, Mb3Cas12a exhibits high efficiency at target sites with a simpler, relaxed TTV PAM which is less restrictive than canonical TTTV LbCas12a AsCas12a. optimize Mb3Cas12a, develop an efficient single transcription unit (STU) system by refining linker between CRISPR RNA (crRNA), nuclear localization signal (NLS), direct repeat (DR). This optimized enables precise rice, for fine-tuning gene expression promoter regions. Further, introduced Arginine (R) substitutions Aspartic acid (D) 172, Asparagine (N) 573, Lysine (K) 579 creating two temperature-tolerant variants: Mb3Cas12a-R (D172R) Mb3Cas12a-RRR (D172R/N573R/K579R). These variants demonstrate significantly improved lower temperatures (22 °C 28 °C) rice cells, showing best performance. We extend this approach developing STU systems maize tomato, achieving biallelic mutants targeting or multiple genes T

Language: Английский

Citations

0