Jinjie Zhu

Upstream open reading frames (uORFs) are pervasive cis-regulatory elements in the 5′untranslated regions (UTRs) of eukaryotic mRNAs that modulate translation of downstream main open reading frames (mORFs) by shaping ribosome scanning, initiation, and reinitiation. By operating downstream of transcription, uORFs enable fine-scale control of protein abundance, offering a distinctive regulatory layer for precise tuning of gene expression in plants. Recent advances in ribosome profiling, translation initiation-site mapping, peptidomics, computational annotation, and genome editing have expanded the discovery, functional validation, and targeted manipulation of plant uORFs. In this review, we summarize current understanding of uORF-mediated translational regulation, discuss computational and experimental approaches for candidate identification and functional validation, and highlight genome-editing strategies used to modify endogenous uORFs in crops. We further synthesize representative studies in which uORF editing has been used to modulate metabolic traits, plant development, nutritional quality, and responses to biotic and abiotic stresses. Collectively, available evidence suggests that uORF manipulation can support quantitative and context-dependent trait modulation, in some cases preserving basal gene function and reducing growth fitness penalties. We further discuss outstanding challenges, including transcript isoform complexity, context-dependent translational effects, and the need for validation across diverse genetic and environmental backgrounds. Overall, uORF engineering represents a promising strategy for translational fine-tuning of plant gene expression, with potential implications for crop improvement and sustainable agriculture.

An adenine base-editing system was established to precisely modify the sorghum SbALS gene, generating transgene-free mutant plants. These plants exhibit strong herbicide resistance, with no significant differences in agronomic traits, providing valuable germplasm for herbicide-resistance breeding in sorghum.

Base editing technologies can improve crops, but their efficiency in maize remains suboptimal. This study attempts to overcome these limitations by examining optimized cytosine and adenine base editors (CBEs and ABEs), namely evoAPOBEC1, evoFERNY, evoCDA1, TadA8.20, and TadA8e, for precise genome editing in transient and stable expression maize cells. Employing a seed fluorescence reporter (SFR) system for rapid screening of BE transformants and transgene‐free progenies, we enhanced editing efficiencies and heritability. Notably, TadA8.20 and evoCDA1 attained multiplexed editing efficiencies of up to 100.0% and 79.0% at the tested loci, respectively, with some homozygous and bi‐allelic mutants exceeding 72.4% and 73.7%. Precise editing of ZmACC1/2 (acetyl‐CoA carboxylase) improved herbicide resistance, with ZmACC2 mutants displaying improved performance. This study advances crop genetic engineering by facilitating robust, multi‐locus modifications without altered agronomic performance, enhancing herbicide tolerance in maize. The successful utilization of these BE is a significant step forward in agricultural biotechnology and precision breeding.

Summary: The integration of haploid induction and genome editing, termed HI‐Edit/IMGE, is a promising tool for generating targeted mutations for crop breeding. However, the technical components and stacking suitable for the maize seed industry have yet to be fully characterised and tested. Here, we developed and assessed three HI‐Edit/IMGE maize lines: Edit^Wx, Edit^Sh, and Edit^Wx&Sh, using the haploid inducer CHOI3 and lines engineered using the CRISPR‐Cas9 system targeting the Waxy1 (Wx1) and Shrunken2 (Sh2) genes. We meticulously characterised the HI‐Edit/IMGE systems, focusing on copy numbers and the mutant alleles mtl and dmp, which facilitate haploid induction. Using B73 and six other parental lines of major commercial varieties as recipients, HI‐Edit/IMGE demonstrated maternal haploid induction efficiencies ranging from 8.55% to 20.89% and targeted mutation rates between 0.38% and 1.46%. Comprehensive assessment verified the haploid identification, target gene editing accuracy, genome background integrity, and related agronomic traits. Notably, Edit^Wx&Sh successfully combined distinct CRISPR‐Cas9 systems to induce multiple desired mutations, highlighting the potential of HI‐Edit/IMGE in accelerating the integration of edited traits into commercial maize varieties. Our findings underscore the importance of meticulous Cas9 copy number characterisation and highlight potential challenges related to somatic chimerism. We also validated the performance of single‐cross haploids derived using the HI‐Edit/IMGE process. Our results confirm the industrial applicability of generating targeted mutations through pollination and provide critical insights for further optimising this technology.

CRISPR/Cas12f proteins belonging to the Type V-F family are reported to be only 1/4 to 1/3 the size of Cas9 protein molecules, providing a significant advantage in viral vector delivery. However, the CRISPR/Cas12f system for gene editing in plants has been reported to have lower editing activity, limiting its broader application in plant research. In this study, we compared the editing activities of OsCas12f, SpCas12f, and UnCas12f in three different systems: in vitro digestion, yeast, and transient expression in maize protoplasts. The results showed that the editing activities of OsCas12f and SpCas12f proteins were comparable in terms of in vitro digestion of Cas12f/sgRNA complexes, while no substrate digestion activity was detected for UnCas12f. In the yeast mutant eGFP expression restoration assay, OsCas12f exhibited an editing efficiency of over 95% at the two tested loci, which was comparable to Cas12i.3. On the other hand, SpCas12f achieved editing efficiencies of 1.63% and 3.20% at the two sites, respectively, representing the next highest effect. However, UnCas12f showed minimal editing activity. Furthermore, by transiently expressing maize protoplasts, we compared the editing efficiencies of OsCas12f and SpCas12f at endogenous maize loci. It was found that OsCas12f successfully mediated targeted editing at two loci with editing efficiencies of 2.72% and 1.97%, respectively, while SpCas12f only mediated targeted editing at one locus with an editing efficiency of 1.09%. Deletion of bases was the predominant type of mutation introduced by Cas12f proteins at the target loci, with deletion lengths ranging from -9 to -17 base pairs. These comprehensive results indicate that OsCas12f can serve as a versatile tool for developing plant microgene editors and related technologies.

The Manipulated Genic Male Sterile Maintainer (MGM) system, a next-generation hybrid seed technology, enables efficient production of sortable seeds from genic male sterile (GMS) lines. However, implementing robust MGM systems in commercial maize inbred lines requires stable transformation, a genotype-specific and laborious process. This study aimed to integrate MGM technology into the commercial maize inbred line Z372, developing both GMS and MGM lines. We utilized the MGM line ZC01-3A-7, which contains the MS26ΔE5 editor T-DNA and MGM T-DNA, previously established in the highly transformable ZC01 recipient plants. Through a combination of crossing and backcrossing with Z372, we targeted the fertility gene Ms26 within the Z372 genome for mutation using the in vivo CRISPR/Cas9 activity within the MS26ΔE5 editor T-DNA construct. This approach facilitated precise editing of the Ms26 locus, minimizing linkage drag associated with the Ms26 mutation. Whole-genome SNP analysis achieved a 98.74% recovery rate for GMS and 96.32% for MGM in the BC2F2 generation. Importantly, the Z372-GMS line with the ms26ΔE5 mutation is non-transgenic, avoiding linkage drag and demonstrating production readiness. This study represents a significant advancement in maize breeding, enabling the rapid generation of GMS and MGM lines for efficient hybrid seed production.

For some Cas nucleases, trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection. Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture. Here, we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool. We systematically characterized AsCas12a, LbCas12a, LwaCas13a, and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection. Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input. Using this tool, we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field. Our method, from sample preparation to result readout, could be rapidly and easily deployed in the field. This system could be extended to other crop pathogens, including those that currently lack a detection method and have metabolite profiles that make detection challenging. This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping, transgene detection, and qualitative detection of gene expression in the field.

Establishing a rapid, accurate, high-throughput and easily implementable genotyping method is highly desirable for functional genomics, genetic improvement and mutant screening. Here, we describe a convenient and inexpensive technique for genotyping using the targeted DNA endonuclease activity of Cas9 or Cas9NG ribonucleoproteins complex (sgRNA/Cas9-RNP or sgRNA/Cas9NG-RNP). In this study, Cas9 and Cas9NG protein purified from E. coli extract was assembled with in vitro transcribed single guide RNA (sgRNA)or enhanced sgRNA (esgRNA) as an assembled ribonucleoprotein (RNP) complex to fulfill the targeted endonuclease activity on the PCR amplicons of ZmWx exon 7. The restriction profiles can be converted into genotyping results of wildtype, homozygous or heterozygous mutant, respectively. Our data showed that ZmWx gene-edited mutants can be genotyped rapidly and efficiently by the sgRNA-optimized system esgRNA/Cas9. The reaction component optimization data suggested that 500 ng of DNA substrates could be cleavaged completely by incubating with 1 µg 1:1 molar ratio of esgRNA/Cas9 ribonucleoproteins for 30 minutes at 37°C, or by 4 µg 1:1 molar ratio of esgRNA/Cas9NG ribonucleoproteins for 4 hours at 37°C. Expanding the targeting flexibility of mutant detection via esgRNA/Cas9NG indicated that Cas9NG variant might recognize relax NG PAM (protospacer-adjacent-motif, PAM) at the expense of decreasing restriction activity, which is necessary to improve the activity of esgRNA/Cas9NG by further optimization. Therefore, the establishment and application of esgRNA/Cas9 based PCR/RNP technique provides an easy, simple and low-cost approach to genotyping in functional genomics, molecular breeding and mutant screening. In addition, our in vitro data on esgRNA/Cas9NG has certain and significant reference value for developing it into in vivo genome editing studies.