Tuning plant phenotypes by precise, graded downregulation of gene expression

Xue, Chenxiao; Qiu, Fengti; Wang, Yuxiang; Li, Boshu; Zhao, Kevin T.; Chen, Kunling; Gao, Caixia

doi:10.1038/s41587-023-01707-w

Cited by 45 publications

(16 citation statements)

References 44 publications

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“…In addition to introducing nucleotide changes, PE can be harnessed for in planta targeted mutagenesis (Xu et al, 2021b) and sitespecific gene regulation in plants (Shi et al, 2023;Tang and Zhang, 2023). By precisely incorporating designed (known) mutations into non-coding regulatory regions or promoter elements or upstream ORFs (Liu et al, 2021a;Rodr ıguez-Leal et al, 2017;Shi et al, 2023;Tang and Zhang, 2023;Xing et al, 2020;Xue et al, 2023;Zeng et al, 2020;Zhang et al, 2018) Wang et al, 2021b;Xing et al, 2020;Xue et al, 2023). Moreover, the PE system can be used to tag endogenous proteins with visible or antigenic markers for in situ study of protein localization, functioning and interaction (Choi et al, 2022b;Li et al, 2023b;Wang et al, 2021a;Zong et al, 2022), though it requires substantial improvement for routine applications.…”

Section: A Recommended Experimental Design For Applying Pe Techniques...mentioning

confidence: 99%

Prime editing: Mechanism insight and recent applications in plants

Vu,

Nguyen,

Kim

et al. 2023

Plant Biotechnology Journal

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

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Section: A Recommended Experimental Design For Applying Pe Techniques...mentioning

confidence: 99%

Prime editing: Mechanism insight and recent applications in plants

Vu,

Nguyen,

Kim

et al. 2023

Plant Biotechnology Journal

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“…By contrast, base editing provides an efficient, precise means to achieve base mutations in target genes without creating DNA double-strand breaks nor requiring the addition of DNA repair templates. Therefore, base editing, dual-base editor in particular, has become an important means of directed evolution of endogenous functional genes, discovery of new beneficial variants, and fine-tuning of gene expression [ 5 , 45 , 46 ].…”

Section: Discussionmentioning

confidence: 99%

Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture

Wang,

et al. 2024

Genome Biol

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Background CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research and crop genetic improvement. However, the editing efficiency of base editors at different targets varies greatly. Results Here, we develop a set of highly efficient base editors in cotton plants. GhABE8e, which is fused to conventional nCas9, exhibits 99.9% editing efficiency, compared to GhABE7.10 with 64.9%, and no off-target editing is detected. We further replace nCas9 with dCpf1, which recognizes TTTV PAM sequences, to broaden the range of the target site. To explore the functional divergence of TERMINAL FLOWER 1 (TFL1), we edit the non-coding and coding regions of GhTFL1 with 26 targets to generate a comprehensive allelic population including 300 independent lines in cotton. This allows hidden pleiotropic roles for GhTFL1 to be revealed and allows us to rapidly achieve directed domestication of cotton and create ideotype germplasm with moderate height, shortened fruiting branches, compact plant, and early-flowering. Further, by exploring the molecular mechanism of the GhTFL1L86P and GhTFL1K53G+S78G mutations, we find that the GhTFL1L86P mutation weakens the binding strength of the GhTFL1 to other proteins but does not lead to a complete loss of GhTFL1 function. Conclusions This strategy provides an important technical platform and genetic information for the study and creation of ideal plant architecture.

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“…The ability to fine‐tune gene expression level is critical for obtaining and analyzing quantitative phenotypic changes (Xue et al., 2023). We showed that the HSP18.2 IGE system exhibited higher Cas9 expression levels than the HSE‐COR15A system in response to heat‐shock treatments.…”

Section: Discussionmentioning

confidence: 99%

Temporally gene knockout using heat shock–inducible genome‐editing system in plants

Liang,

Wei,

et al. 2023

The Plant Genome

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Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated nuclease 9 (Cas9) has emerged as a powerful tool to generate targeted loss‐of‐function mutations for functional genomic studies. As a next step, tools to generate genome modifications in a spatially and temporally precise manner will enable researchers to further dissect gene function. Here, we present two heat shock–inducible genome‐editing (IGE) systems that efficiently edit target genes when the system is induced, thus allowing us to target specific developmental stages. For this conditional editing system, we chose the natural heat‐inducible promoter from heat‐shock protein 18.2 (HSP18.2) from Arabidopsis thaliana and the synthetic heat–inducible promoter heat shock–response element HSE‐COR15A to drive the expression of Cas9. We tested these two IGE systems in Arabidopsis using cyclic or continuous heat‐shock treatments at the seedling and bolting stages. A real‐time quantitative polymerase chain reaction analysis revealed that the HSP18.2 IGE system exhibited higher Cas9 expression levels than the HSE‐COR15A IGE system upon both cyclic and continuous treatments. By targeting brassinosteroid‐insensitive 1 (BRI1) and phytoene desaturase (PDS), we demonstrate that both cyclic and continuous heat inductions successfully activated the HSP18.2 IGE system at the two developmental stages, resulting in highly efficient targeted mutagenesis and clear phenotypic outcomes. By contrast, the HSE‐COR15A IGE system was only induced at the seedling stage and was less effective than the HSP18.2 IGE system in terms of mutagenesis frequencies. The presented heat shock–IGE systems can be conditionally induced to efficiently inactivate genes at any developmental stage and are uniquely suited for the dissection and systematic characterization of essential genes.

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Tuning plant phenotypes by precise, graded downregulation of gene expression

Cited by 45 publications

References 44 publications

Prime editing: Mechanism insight and recent applications in plants

Prime editing: Mechanism insight and recent applications in plants

Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture

Temporally gene knockout using heat shock–inducible genome‐editing system in plants

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