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Summary: 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 in plants. To enhance the utility of Cas12a, we investigate three Cas12a orthologs—Mb3Cas12a, PrCas12a, and HkCas12a—in plants. Protospacer adjacent motif (PAM) requirements, editing efficiencies, and editing profiles are compared in rice. Among these orthologs, Mb3Cas12a exhibits high editing efficiency at target sites with a simpler, relaxed TTV PAM which is less restrictive than the canonical TTTV PAM of LbCas12a and AsCas12a. To optimize Mb3Cas12a, we develop an efficient single transcription unit (STU) system by refining the linker between Mb3Cas12a and CRISPR RNA (crRNA), nuclear localization signal (NLS), and direct repeat (DR). This optimized system enables precise genome editing in rice, particularly for fine‐tuning target gene expression by editing promoter regions. Further, we introduced Arginine (R) substitutions at Aspartic acid (D) 172, Asparagine (N) 573, and Lysine (K) 579 of Mb3Cas12a, creating two temperature‐tolerant variants: Mb3Cas12a‐R (D172R) and Mb3Cas12a‐RRR (D172R/N573R/K579R). These variants demonstrate significantly improved editing efficiency at lower temperatures (22 °C and 28 °C) in rice cells, with Mb3Cas12a‐RRR showing the best performance. We extend this approach by developing efficient Mb3Cas12a‐RRR STU systems in maize and tomato, achieving biallelic mutants targeting single or multiple genes in T0 lines cultivated at 28 °C and 25 °C, respectively. This study significantly expands Cas12a's targeting capabilities in plant genome editing, providing valuable tools for future research and practical applications. [ABSTRACT FROM AUTHOR]

To meet the growing demand for food from human being, the study on editing important gene-coding regions to produce beneficial traits is countless. Transcriptional regulation is crucial for controlling gene expression, including the control of important agronomic traits in crops. The interaction between cis-acting elements and trans-acting factors determines the spatiotemporal expression patterns and levels of genes, and transcription factor binding sites and enhancers etc. play an important role in those pathways. What's more, the promoter, as the largest and most important cis-acting element, can be modified to alter the expression patterns of target genes, and create beneficial traits in crops, which is an effective strategy different from coding region editing breeding. There are two applications of promoter editing strategies. One is directed modification to generate specific beneficial traits, and the other is to randomly generate new alleles within specific promoter regions, followed by phenotypic selection, also producing new genetic variation resources. In this review, we mainly discuss the application of promoter editing in crops, including increasing yield, improving quality, and enhancing tolerance to biotic and abiotic stresses. It aims to focus on the cutting-edge precision breeding of crops, providing research ideas and theoretical references for the development and application of promoter gene editing technology. [ABSTRACT FROM AUTHOR]

Abstract Gene expression levels in rice (Oryza sativa L.) and other plant species are determined by the promoters, which directly control phenotypic characteristics. As essential components of genes, promoters regulate the intensity, location, and timing of gene expression. They contain numerous regulatory elements and serve as binding sites for proteins that modulate transcription, including transcription factors and RNA polymerases. Genome editing can alter promoter sequences, thereby precisely modifying the expression patterns of specific genes, and ultimately affecting the morphology, quality, and resistance of rice. This paper summarizes research on rice promoter editing conducted in recent years, focusing on improvements in yield, heading date, quality, and disease resistance. It is expected to inform the application of promoter editing and encourage further research and development in crop genetic improvement with promote.

Gene expression levels in rice (Oryza sativa L.) and other plant species are determined by the promoters, which directly control phenotypic characteristics. As essential components of genes, promoters regulate the intensity, location, and timing of gene expression. They contain numerous regulatory elements and serve as binding sites for proteins that modulate transcription, including transcription factors and RNA polymerases. Genome editing can alter promoter sequences, thereby precisely modifying the expression patterns of specific genes, and ultimately affecting the morphology, quality, and resistance of rice. This paper summarizes research on rice promoter editing conducted in recent years, focusing on improvements in yield, heading date, quality, and disease resistance. It is expected to inform the application of promoter editing and encourage further research and development in crop genetic improvement with promote. [ABSTRACT FROM AUTHOR]

Summary: Heading date (or flowering time) is a key agronomic trait that affects seasonal and regional adaption of rice cultivars. An unoptimized heading date can either not achieve a high yield or has a high risk of encountering abiotic stresses. There is a strong demand on the mild to moderate adjusting the heading date in breeding practice. Genome editing is a promising method which allows more precise and faster changing the heading date of rice. However, direct knock out of major genes involved in regulating heading date will not always achieve a new germplasm with expected heading date. It is still challenging to quantitatively adjust the heading date of elite cultivars with best adaption for broader region. In this study, we used a CRISPR‐Cas9 based genome editing strategy called high‐efficiency multiplex promoter‐targeting (HMP) to generate novel alleles at cis‐regulatory regions of three major heading date genes: Hd1, Ghd7 and DTH8. We achieved a series of germplasm with quantitative variations of heading date by editing promoter regions and adjusting the expression levels of these genes. We performed field trials to screen for the best adapted lines for different regions. We successfully expanded an elite cultivar Ningjing8 (NJ8) to a higher latitude region by selecting a line with a mild early heading phenotype that escaped from cold stress and achieved high yield potential. Our study demonstrates that HMP is a powerful tool for quantitatively regulating rice heading date and expanding elite cultivars to broader regions. [ABSTRACT FROM AUTHOR]

Summary: The CRISPR‐Cas‐based genome editing field in plants is expanding rapidly. Editing plant promoters to obtain cis‐regulatory alleles with altered expression levels or patterns of target genes is a highly promising topic. However, primarily used CRISPR‐Cas9 has significant limitations when editing noncoding sequences like promoters, which have unique structures and regulatory mechanisms, including A‐T richness, repetitive redundancy, difficulty in identifying key regulatory regions, and a higher frequency of DNA structure, epigenetic modification, and protein binding accessibility issues. Researchers urgently require efficient and feasible editing tools and strategies to address these obstacles, enhance promoter editing efficiency, increase diversity in promoter polymorphism, and, most importantly, enable 'non‐silent' editing events that achieve precise target gene expression regulation. This article provides insights into the key challenges and references for implementing promoter editing‐based research in plants. [ABSTRACT FROM AUTHOR]

Genome-edited plants, endowed with climate-smart traits, have been promoted as tools for strengthening resilience against climate change. Successful plant gene editing (GE) requires precise regulation of the GE machinery, a process controlled by the promoters, which drives its transcription through interactions with transcription factors (TFs) and RNA polymerase. While constitutive promoters are extensively used in GE constructs, their limitations highlight the need for alternative approaches. This review emphasizes the promise of tissue/organ specific as well as inducible promoters, which enable targeted GE in a spatiotemporal manner with no effects on other tissues. Advances in synthetic biology have paved the way for the creation of synthetic promoters, offering refined control over gene expression and augmenting the potential of plant GE. The integration of these novel promoters with synthetic systems presents significant opportunities for precise and conditional genome editing. Moreover, the advent of bioinformatic tools and artificial intelligence is revolutionizing the characterization of regulatory elements, enhancing our understanding of their roles in plants. Thus, this review provides novel insights into the strategic use of promoters and promoter editing to enhance the precision, efficiency and specificity of plant GE, setting the stage for innovative crop improvement strategies., (© 2025 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Modulation of stomatal development and movement is a promising approach for creating water-conserving plants. Here, we identified and characterized the PagHCF106 gene of poplar (Populus alba × Populus glandulosa). The PagHCF106 protein localized predominantly to the chloroplast and the PagHCF106 gene exhibited a tissue-specific expression pattern. Overexpression of PagHCF106 rescued the reduced stomatal aperture and increased drought resistance of the hcf106 Arabidopsis mutant. Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9-mediated genome editing introduced mutations in the core region of the PagHCF106 promoter, which was required for its activity, as confirmed by the GUS staining assay. Mutation of the PagHCF106 promoter reduced stomatal opening and water loss in poplar. In addition, the genome-edited poplar lines accumulated high levels of hydrogen peroxide in guard cells. Taken together, these results suggest that PagHCF106 negatively regulates drought stress tolerance by modulating stomatal aperture, which might be associated with reactive oxygen species production. We propose that modification of the PagHCF106 promoter might be an efficient strategy for enhancing drought stress tolerance in poplar., (© The Author(s) 2025. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Summary: The low efficiency of genetic transformation and gene editing across diverse cultivars hinder the broad application of CRISPR technology for crop improvement. The development of virus‐based methods of CRISPR‐Cas system delivery into the plant cells holds great promise to overcome these limitations. Here, we perform direct inoculation of wheat leaves with the barley stripe mosaic virus (BSMV) transcripts to deliver guide RNAs (sgRNA) into the Cas9‐expressing wheat. We demonstrate that wheat inoculation with the pool of BSMV‐sgRNAs could be used to generate heritable precise deletions in the promoter region of a transcription factor and to perform multiplexed editing of agronomic genes. We transfer the high‐expressing locus of Cas9 into adapted spring and winter cultivars by marker‐assisted introgression and use of the BSMV‐sgRNAs to edit two agronomic genes. A strategy presented in our study could be applied to any adapted cultivar for creating new cis‐regulatory diversity or large‐scale editing of multiple genes in biological pathways or QTL regions, opening possibilities for the effective engineering of crop genomes, and accelerating gene discovery and trait improvement efforts. [ABSTRACT FROM AUTHOR]

The percentage of chalky grains in WYJ30 was 12.1%, whereas that of WYJ30-T3-1, WYJ30-T3-2 and WYJ30-T3-3 was 10.0%, 7.8% and 4.5%, respectively (Figure 1f). Keywords: rice; appearance quality; grain shape; chalkiness; promoter editing; regulatory element EN rice appearance quality grain shape chalkiness promoter editing regulatory element 1305 1307 3 06/22/23 20230701 NES 230701 Rice, an important food crop consumed by more than half of the world's population, is also a tradeable commodity. Unlabelled WYJ30-T3-1, WYJ30-T3-2 and WYJ30-T3-3 promoter probes were used (100-fold) for the competition in (p). Compared with WYJ30, the mutants WYJ30-T3-1, WYJ30-T3-2 and WYJ30-T3-3 produced more slender grains (Figure 1c, e). [Extracted from the article]

Chromatin interactions create spatial proximity between distal regulatory elements and target genes in the genome, which has an important impact on gene expression, transcriptional regulation, and phenotypic traits. To date, several methods have been developed for predicting gene expression. However, existing methods do not take into consideration the effect of chromatin interactions on target gene expression, thus potentially reducing the accuracy of gene expression prediction and mining of important regulatory elements. In this study, we developed a highly accurate deep learning-based gene expression prediction model (DeepCBA) based on maize chromatin interaction data. Compared with existing models, DeepCBA exhibits higher accuracy in expression classification and expression value prediction. The average Pearson correlation coefficients (PCCs) for predicting gene expression using gene promoter proximal interactions, proximal-distal interactions, and both proximal and distal interactions were 0.818, 0.625, and 0.929, respectively, representing an increase of 0.357, 0.16, and 0.469 over the PCCs obtained with traditional methods that use only gene proximal sequences. Some important motifs were identified through DeepCBA; they were enriched in open chromatin regions and expression quantitative trait loci and showed clear tissue specificity. Importantly, experimental results for the maize flowering-related gene ZmRap2.7 and the tillering-related gene ZmTb1 demonstrated the feasibility of DeepCBA for exploration of regulatory elements that affect gene expression. Moreover, promoter editing and verification of two reported genes (ZmCLE7 and ZmVTE4) demonstrated the utility of DeepCBA for the precise design of gene expression and even for future intelligent breeding. DeepCBA is available at http://www.deepcba.com/ or http://124.220.197.196/., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Developing nutritious rice with a higher yield is one approach to alleviating the problem of micronutrient deficiency in developing countries, especially human malnutrition involving zinc and iron (Fe) deficiency, and achieving better adoption. The transport of micronutrients such as Fe and Zn is mainly regulated via the nicotianamine synthase ( OsNAS ) gene family, whereas yield is a complex trait that involves multiple loci. Genome editing via CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, focusing on the OsNAS2 promoter, particularly the deletion of the cis -regulatory element ARR1AT at position -933, was conducted for an enhanced accumulation of Zn in the grain and per plant. The results showed that our promoter editing increased Zn concentration per plant. Evidence also showed that an improved spikelet number per main panicle led to increased grain per plant. The traits were inherited in "transgene-free" and homozygous plant progenies. Further investigation needs to be conducted to validate trait performance under field conditions and elucidate the cause of the spikelet increase., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ludwig, Dueñas, Arcillas, Macalalad-Cabral, Kohli, Reinke and Slamet-Loedin.)