CARBON Newsletter (24 September 2024) - Your Latest News About CRISPR in AgroBio

Top picks

• Spanish researchers have used CRISPR-Cas multiplexing to target and mutate wheat gliadin genes, which are responsible for gluten-related reactions. By designing eight guide RNAs, they generated 59 modified lines, 20 of which showed mutations. Analysis revealed a 97.7% reduction in gluten content, with mutations passed to offspring. Crossing these lines with others deficient in α-gliadins advances the development of gluten-free wheat varieties.
• Korean scientists have optimised prime editing (PE) for dicots by combining an RNA chaperone, engineered prime editing guide RNAs, and a viral replicon system, achieving up to 9.7% efficiency at the callus stage. Heritable PE alleles were successfully transmitted in tomatoes and Arabidopsis, with up to 38.2% of transformants containing desired edits. This advancement significantly improves PE accuracy, specificity, and multiplexing in dicots, offering transformative potential for plant research. The research journey behind the breakthrough is detailed by the original authors in this post.

Technical advances

• Marking a first for ferns, Australian researchers have applied CRISPR-Cas9 to Ceratopteris richardii gametophytes. By using specific promoters, the system successfully edited genes such as CrSAL1 and CrPDS, inducing phenotypic changes like reduced stomatal conductance and altered photosynthesis. Overexpression of CrSAL1 enhanced stomatal traits and ROS production. This protocol offers a novel tool for fern functional genomics, with potential applications in evolutionary biology and agriculture. Earlier this year, Chinese researchers were the first to use CRISPR gene-editing in callus from C. richardii.
• Researchers in Japan have developed a DNA-free genome editing method for soybeans using the in planta bombardment-ribonucleoprotein (iPB-RNP) system, targeting shoot apical meristem cells with CRISPR-Cas9 complexes. This approach bypasses traditional tissue culture and successfully induces heritable mutations in allergenic and other genes, offering a practical method for plant regeneration and precise editing in dicotyledonous crops without introducing foreign DNA.
• Other Japanese scientists present another approach to in-plant particle bombardment (iPB-RNP) in melon in a paper that has not yet been reviewed. The method was used to create CmACO1 mutants in melon, resulting in extended shelf life due to reduced ethylene production. This method bypasses cell culture, addressing genotype dependency and somatic variation issues, offering promising applications for commercial breeding in melon and other Cucurbitaceae species.
• American researchers have developed RNA virus-mediated delivery of single-guide RNAs (sgRNAs) for gene editing in Cas9-expressing tomato plants. The novel approach overcomes tissue culture limitations by inducing gene-edited shoots from somatic cells near apical meristems. Additionally, the co-delivery of sgRNAs and cytokinin biosynthesis genes boosted shoot induction, enabling efficient, heritable gene edits in tomatoes, thus advancing virus-based genome editing in dicot crops.
• CRISPR-Cas13a was integrated into a new detection method for plant miRNA, Cas13a-SATA, overcoming challenges posed by 3’-terminal modifications. The strategy combines DNA self-assembly and transcription amplification to activate Cas13a's trans-cleavage activity, generating a fluorescence signal proportional to miRNA levels. This method detected plant miR156a with high sensitivity (LOD: 3.8 fM) and successfully quantified miR156a in Arabidopsis and maise, highlighting its potential for accurate miRNA analysis in biological samples.
• In a new study, researchers have developed CRISPR-dCas13-based tools for precise m⁶A RNA editing in plants, enabling targeted methylation or demethylation of transcripts. By modifying m6A sites on GhECA1 and GhDi19, these editors reveal opposing regulatory effects. Targeted methylation increases root length and drought resistance, highlighting their potential for crop improvement.
• American scientists have investigated the heritability of CRISPR-Cas12a-mediated on-target and off-target edits in soybeans, revealing that careful crRNA design significantly reduces off-target mutations. On-target edits were inherited in 80% of T1 plants, while off-target edits occurred with "promiscuous" crRNAs but not "unique" ones. The findings confirm that selecting precise crRNAs ensures the propagation of plants with desired edits and minimal unintended changes.
• In a paper still under review, researchers have developed PREMJ, a CRISPR-Cas-based tool for precision gene replacement in plants using microhomology-mediated end joining (MMEJ). PREMJ, combined with an inhibitor of nonhomologous end-joining, improved gene replacement efficiency in tomato, lettuce, and cabbage protoplasts. While challenges remain in regenerating edited plants, this approach shows promise for improving gene editing in species capable of efficient delivery and regeneration.

Disease and stress control

• Knockout mutants of OsWRKY70 generated by CRISPR-Cas9 have revealed the gene's dual role in rice stress responses. Mutants showed enhanced resistance to Magnaporthe oryzae, with increased ROS production and defence gene activation. However, cold tolerance was impaired, linked to reduced antioxidant activity and downregulation of cold-responsive genes. OsWRKY70 also regulates OsbHLH6 in the JA pathway, providing new strategies for stress-tolerant crop breeding.
• As pests develop resistance to Bacillus thuringiensis (Bt) insecticidal proteins, the long-term effectiveness of Bt crops is threatened. Understanding Bt resistance mechanisms is crucial in managing this challenge. This study confirms that the T92C mutation in the HaTSPAN1 gene causes dominant resistance to Cry1Ac in Helicoverpa armigera, with transgenic strains created using the piggyBac gene-editing system showing 82-fold resistance. The findings highlight the mutation's role in Bt resistance and demonstrate the potential of piggyBac-mediated transformation for investigating resistance genes in other pests.

Agronomic traits

• Researchers in Korea have used CRISPR-Cas9 ribonucleoproteins to efficiently edit the CaPAD1 gene in three pepper cultivars (Capsicum annuum) to induce parthenocarpy, targeting seedless fruit production under high-temperature stress. Two guide RNAs with high in vivo efficiency were identified, offering a promising strategy for developing climate-resilient, seedless pepper varieties through precise molecular breeding.
• Mutations in potato starch-branching enzyme (SBE) genes generated by CRISPR-Cas9 revealed that modified starches had higher gelatinisation temperatures and retrograded faster than wild-type. While all native starches resisted digestion, prolonged retrogradation reduced digestibility in SBE-modified lines, suggesting that altering SBE genes can enhance starch resistance, potentially improving dietary fibre content in potatoes.
• CRISPR-Cas9 knockout of ClDUF21 in watermelon has revealed its role in dwarfism by modulating brassinosteroid synthesis through interaction with ClDWF1. Mutant plants exhibited a pronounced dwarf phenotype. Similar results were achieved in cucumber, where CRISPR targeting of the homologous gene CsDUF21 confirmed its role in plant dwarfism. These findings provide insights into the genetic regulation of compact growth, which is beneficial for intensive farming.
• Knocking out the Glu-B gene in rice using CRISPR-Cas9 reduced glutelin (GLU) content and increased levels of globulins, albumins, and prolamins. This mutation also resulted in lower starch and amylose levels, looser starch granules, and higher grain chalkiness. The study highlights Glu-B's key role in regulating protein composition, offering a path to breed rice with enhanced nutritional profiles for GLU-sensitive individuals.
• CRISPR-Cas9 was used to knock out the GhMYB201 gene in cotton, revealing its role in fibre elongation. Loss of GhMYB201 resulted in shorter, coarser fibres, highlighting its influence on both cell wall loosening and very-long-chain fatty acid biosynthesis. This transcription factor regulates the expression of genes involved in these processes, enhancing our understanding of fibre quality control in cotton.
• Chinese researchers have used CRISPR-Cas9 to target SlMTC, an m6A methylation enzyme, in tomatoes. Knockout of S1MTC led to reduced seed size, slower seedling growth, and weakened salt tolerance compared to wild-type plants. Mutants showed downregulated stress-related and auxin-related genes, impairing growth and stress responses. SlMTC interacted with SlMTA, another MT-A70 family protein, suggesting a complex regulatory mechanism in plant development and salt stress adaptation.
• CRISPR-Cas9 has been used to generate OsWOX13 overexpression (OsWOX13-ov) and knockout (oswox13-ko) rice lines, which flowered 10 days earlier and 4–6 days later than wild-type, respectively. qRT-PCR suggests OsWOX13 regulates drought escape during flowering via the OsbZIP23 pathway, affecting genes like Ghd7 and Hd3a. Targeting OsWOX13 could enhance stress resilience and optimise flowering time for improved rice productivity.
• Researchers have used CRISPR-Cas9 to knock out the SlXTH5 gene in tomatoes, revealing its role in fruit ripening. Mutant plants showed accelerated ripening, higher ethylene accumulation, and improved fruit firmness without changes in size or weight. Enhanced pathogen resistance and longer shelf life were linked to thicker cell walls, highlighting SlXTH5's function in cell wall metabolism and softening.
• Researchers in Japan have used CRISPR-Cas9 to target two enzymes in the fatty acid beta-oxidation pathway of Euglena gracilis to alter wax ester production. Genetic mutations modified the wax ester chain length, with a triple-knockout mutant producing esters with acyl chains two carbons shorter than the wild type. This method provides a stable approach for modifying wax ester composition, enhancing microalgal biofuel potential.

Industry

• Pairwise, a leader in gene editing for agriculture, has secured $40 million in Series C funding led by Deerfield Management, with new support from Corteva Catalyst. The investment will expand Pairwise's innovative crop development, such as seedless berries and thornless plants, and enhance its Fulcrum™ Platform, which uses CRISPR to tailor plant traits. The joint venture with Corteva will focus on climate-resilient, high-yield crops, reinforcing the promise of gene editing in agriculture.
• Corteva and Pairwise have announced a partnership to enhance the development of gene-edited crops aimed at improving climate resilience and increasing productivity for food, fuel, and fibre. Corteva invested $25 million in Pairwise to accelerate gene editing innovations for various crops, including corn, soy, and canola. The joint venture will leverage both companies' expertise to create crops better suited to extreme weather conditions and higher yields, benefiting both farmers and consumers.

Commentaries and opinions

• An editorial in Frontiers in Plant Science discusses the critical role of advanced plant biotechnology and genetics in enhancing global food security amidst challenges like climate change and economic instability. The focus is on modern techniques such as CRISPR, omics technologies, and genome-wide association studies (GWAS), which offer novel solutions for crop resilience, yield improvement, and sustainability in agriculture.
• An article in The Conversation describes how gene-edited plants are moving from labs to homes, offering innovations like prickle-free roses and antioxidant-rich purple tomatoes. CRISPR and DNA modification enable targeted mutations and transgenic organisms, creating new varieties tailored for gardeners and consumers. While this technology presents exciting possibilities, including bioluminescent plants and compact crops, concerns about ecological risks and market regulation highlight the need for cautious oversight as these plants become more widely accessible.
• According to an article in AgTechNavigator, plant breeders are calling for unified global regulations on genome-edited crops to enhance food security in the face of climate change. A survey by the International Seed Federation highlights the need for science-based, harmonised policies to accelerate innovation, reduce regulatory fragmentation, and promote resilient crop varieties. Such measures could ensure more efficient breeding and support sustainability efforts in agriculture worldwide.

Reviews

• Recent advances of CRISPR-based genome editing for enhancing staple crops. This review examines the transformative potential of CRISPR-Cas technology in addressing global food security challenges by enhancing crop resilience, yield, and quality through precise genetic modifications.
• Genome editing in angiosperm chloroplasts: targeted DNA double-strand break and base editing. This review discusses genome editing methods targeting the chloroplast genome, highlighting their advantages, limitations, and the current reliance on protein-based enzymes over CRISPR-Cas systems due to challenges in delivering guide RNAs into chloroplasts.
• Genome editing for improvement of biotic and abiotic stress tolerance in cereals. This review focuses on how CRISPR/Cas9 genome editing might enhance cereals' agronomic qualities in the face of climate change, providing important insights for future applications.
• Genetic, molecular and physiological crosstalk during drought tolerance in maise (Zea mays): pathways to resilient agriculture. This comprehensive review covers quantitative trait loci mapping, genome-wide association studies, essential genes and functions, CRISPR-Cas applications, transcription factors, physiological responses, and signalling pathways, offering a nuanced understanding of intricate mechanisms involved in maise drought tolerance.
• Physiological and Molecular Mechanisms of Rice Tolerance to Salt and Drought Stress: Advances and Future Directions. This review outlines how rice stress tolerance can be significantly enhanced by CRISPR-Cas9 gene editing of genetic pathways of crucial regulators like transcription factors such as DREB, NAC, and bZIP, as well as plant hormones like ABA and GA.
• Revolutionising soybean genomics: How CRISPR and advanced sequencing are unlocking new potential. This review discusses advances in soybean genomics, focusing on genome sequencing, CRISPR-Cas9 editing, and genetic resources that enable improved traits such as yield, nutrition, and stress resistance.
• Advances in breeding, biotechnology, and nanotechnological approaches to combat sheath blight disease in rice. This review focuses on recent advancements in combating rice sheath blight disease, including advanced breeding, transgenic methods, CRISPR-Cas9 genome editing, and nanotechnological interventions.
• Advances in plant pathogen detection: integrating recombinase polymerase amplification with CRISPR/Cas systems. This review summarises recent advances in RPA-CRISPR/Cas12a technology for plant pathogen detection, highlighting its rapid, sensitive, and field-portable capabilities while addressing challenges like primer design and operational efficiency.
• CRISPR/Cas in Grapevine Genome Editing: The Best Is Yet to Come. This review discusses the development, applications, challenges, and prospects of CRISPR-Cas technology for genome editing in grapevine, highlighting its potential to accelerate genetic improvement and trait enhancement in this economically important species.