Yubing He

Editing TaBADH1 alone or TaBADH1 and TaBADH2 via CRISPR/Cas9 produced fragrant wheat with up to 11.24-fold higher 2-acetyl-1-pyrroline than the wild type without yield penalty, revealing asymmetric functional redundancy between these two genes and providing elite germplasm for wheat quality improvement.

CRISPR/Cas12i3 belongs to the type V‐I Cas system, characterized by its smaller protein size and less restricted canonical “TTN” protospacer adjacent motif. Developments of Cas12i3‐mediated base editing systems for either C‐to‐T or A‐to‐G transitions will expand the editing scope and enrich the plant base editing toolkits for crop improvement. However, while the Cas12i3‐based cytosine base editor (CBE) only shows very low editing efficiency in plants, its adenine base editor (ABE) has not been documented as yet. Here, we engineered a series of Cas12i3 (5M)‐based CBEs (V0–V5) and ABEs (V0–V5) by fusing a deactivated dCas12i3 (5M) with a transactivation module VP64, a single‐stranded DNA‐binding domain Rad51, or a double‐stranded DNA‐binding domain HMG‐D, or in combinations, and systemically evaluated their performance in rice protoplasts. Our results demonstrated that synergistic combinations of both VP64 and HMG‐D outperformed other architectures and significantly boosted the efficiencies of Cas12i3 (5M)‐based CBE and ABE for C‐to‐T and A‐to‐G base editing and expanded the editing window. In stable lines, in comparison to the non‐fusion control, the optimized Cas12i3 (5M)‐based CBE‐V5 and ABE‐V5 enabled up to 4.78‐ and 3.35‐fold higher editing efficiencies, with the maximum C‐to‐T and A‐to‐G efficiencies reaching 32.35% and 38.24%, respectively, and a higher proportion of homozygous mutants in the T ₀ generation. Furthermore, we generated herbicide‐resistant rice germplasm by using CBE‐V5 and ABE‐V5, demonstrating their potential for precision breeding in crops. Together, here, we report novel Cas12i3 (5M)‐based CBE and ABE that substantially enrich base editing toolkits for improvement of rice and potentially other crops.

A simple, fast, and genotype-independent RUBY-assisted transformation method turns soybean seedlings into genetically modified plants in around three months without laboratory sterile culture.

The CRISPR/Cas9 gene‐editing technology has been widely used in defining gene functions and crop improvement. However, some genes are essential for plant growth and development. Loss‐of‐function homozygous mutations in essential genes lead to plant death or sterility. Mutations in essential genes need to be maintained and propagated in heterozygous plants. CRISPR/Cas9 technology is highly efficient in generating homozygous or bi‐allelic mutations at T0 generation in rice, making it difficult to generate useful genetic materials for essential genes using traditional gene editing technology. In this study, we designed Transgene‐Killer CRISPR (TKC)‐mediated mismatch‐spacer targeting (TKC‐M) to efficiently generate heritable heterozygous mutations in essential genes in rice. Leveraging our earlier transgenic offspring self‐elimination TKC platform, TKC‐M relied on timely self‐elimination of Cas9 and engineered gRNA‐target mismatches to enrich heritable heterozygous or mosaic incomplete‐edited T0 mutants and heterozygous progeny. We found that the sensitivity of targets to spacer mismatch(es) varies. A single‐base mismatch at gRNA positions 11 or 17 yielded abundant heritable heterozygotes in sensitive targets. For insensitive targets, dual mismatches at positions 8 and 15 maximised heritable heterozygotes. Co‐transformation of rice with TKC vectors carrying gRNA without mismatches (G1), gRNA with a mismatch at position 11 (M11) and M8 + M15 spacers, termed TKC‐M Cocktail (TKC‐MC) significantly increased the incomplete‐edited mutant ratio compared with using G1 alone. This work establishes a technical foundation for generating mutant libraries that cover every single gene in a plant genome and for in‐depth research on essential genes.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas)9 genome-editing technology has become a cornerstone for generating knockout mutations in plant functional genomics. To obtain genetically stable CRISPR-edited plants, the removal of exogenous CRISPR constructs through genetic segregation is imperative. However, current transgene-free strategies lack universality and operational simplicity. Here, we developed a modular CRISPR toolkit integrated with the widely applicable visual RUBY marker. This system achieved 100% editing efficiency in three independent CRISPR-Cas9 editing events in rice (cv. Zhonghua 11), enabled rapid visual identification of transgene-free progeny, and may provide a framework for future adaptation of CRISPR vectors to other plant species. Our design significantly accelerates the identification of edited lines while bypassing laborious molecular validation steps.

Summary: A critical step in generating gene‐edited plants is the removal of CRISPR‐related transgenes from T₀ plants and their progenies, a process that is generally time‐consuming and labour‐intensive. We previously reported a Transgene Killer CRISPR (TKC) technology that enables self‐elimination of transgenes after the targeted gene has been edited. However, we observed that a small number of T₁ plants generated by TKC still retained the transgenes. Herein, we first integrated Cas9 and guide RNA (gRNA) with the RUBY reporter gene (RUBY‐CRISPR) to monitor the Cas9/sgRNA expression and track the presence or absence of transgenes in the T₀ generation and its progenies. We then combined the RUBY‐CRISPR unit with several TKC variants to develop four RUBY‐TKC (TKC2) systems including TKC2.1, TKC2.2, TKC2.3 and TKC2.4, to facilitate the elimination of escaped transgene plants. Compared to non‐TKC, TKC alone and RUBY‐CRISPR, our TKC2s were much more efficient in the generation of transgene‐free edited progenies by up to 100% in the T₀ generation. TKC2s not only omit the need for screening of the plants with transgenes in the T₀ generation, but also enable visualisation of the escape of plants with transgenes in the following progenies. The TKC2 systems developed here provide straightforward yet highly effective approaches for the generation of transgene‐free edited plants for either rice functional genomics or genetic improvement, with potential applications in gene editing of other crops.

Regulation of DNA or RNA at the transcriptional, post-transcriptional, and translational levels are key steps in the central dogma of molecular biology. DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs) play pivotal roles in the precise regulation of gene expression in these steps. Both of these two classes of proteins are nucleic acid-binding proteins (NABPs), so they exhibit significant similarity in both sequence and structure. However, traditional methods for identifying NABPs are typically time-consuming, costly, and challenging to scale up. Utilizing deep learning to classify proteins intelligently has emerged as a more efficient solution for these issues. In this study, we propose a phased classification method integrating ESM-2 with a dual-path neural network, called DRBP-EDP. Additionally, a refined approach to dataset construction is designed, resulting in the creation of high-quality protein classification datasets. The results demonstrated that the model achieved strong performance, with 90.03% accuracy in the first stage for classifying NABPs and non-nucleic acid-binding proteins, and 89.56% accuracy in the second stage for classifying DBPs and RBPs. To enhance accessibility and usability, DRBP-EDP has been developed in both executable and web-based versions, which are publicly available at https://doi.org/10.5281/zenodo.14092184 and https://github.com/MuQiang-MQ/DRBP-EDP.

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