CARBON Newsletter (29 October 2024) - Your Latest News About CRISPR in AgroBio

Top picks

• American researchers have successfully used precision adenine base editing to introduce a single amino acid mutation (S264G) in the chloroplast psbA gene, conferring herbicide resistance in Arabidopsis. The method bypasses CRISPR enzymes by employing so-called UDG-TALEDs that are composed of transcription activator-like effectors (TALE)-linked adenine deaminase fused with uracil DNA glycosylase. The UDG-TALEDs are designed to perform precise A-to-G edits while minimising unintended C-to-T conversions in organellar DNA.
• CRISPR-Cas9 knockout of the WUSCHEL-related homeobox (WOX) transcription factor SlWOX4 gene in tomatoes enhances drought tolerance by decreasing water loss, improving stomatal closure, and increasing antioxidant activity and proline accumulation. The edited plants showed lower reactive oxygen species and malondialdehyde levels under drought, suggesting SlWOX4 as a key negative regulator of drought response, with potential utility in breeding drought-resistant tomato varieties.

Technical advances

• American researchers have introduced a virus-mediated approach to deliver CRISPR sgRNAs to Cas9-expressing tomato plants, bypassing tissue culture. Viral sgRNAs targeting axillary meristems, combined with cytokinin biosynthesis gene delivery, successfully generated phenotypically normal, heritable gene-edited shoots. This technique enables efficient gene editing in dicot crops like tomato, potentially accelerating genome editing applications by overcoming traditional tissue culture limitations.
• A new study advances multiplex CRISPR-Cas9 editing in Citrus by optimising tRNA-based sgRNA arrays and carefully selecting promoters to boost editing efficiency. The Arabidopsis UBQ10 and RPS5a promoters proved effective in driving zCas9i expression, while Pol III, UBQ10, and ES8Z promoters reliably enhanced sgRNA array expression. These insights enable more efficient, simultaneous multigene editing in the Carrizo citrange cultivar, offering valuable guidance for CRISPR applications in Citrus and other eudicots.
• A new study reveals that CRISPR-Cas12a-generated 5' overhang DSBs in Arabidopsis thaliana are primarily repaired by local end-joining, often resulting in small deletions. Nanopore sequencing shows that paired DSBs create deletions and occasional inversions. Notably, DSBs flanked by direct repeats exhibit a distinct repair pattern dominated by single-strand annealing recombination, highlighting sequence context as a significant factor in DNA repair pathways.
• A rapid and efficient Agrobacterium tumefaciens-mediated genetic transformation system was developed for the traditional medical plant Descurainia sophia by briefly dipping inflorescences in an Agrobacterium suspension. The system, validated with CRISPR-Cas9 targeting the PDS gene, produced albino mutants within 2.5 months.
• A new study advances CRISPR-Cas12a genome editing in plants by optimising Mb3Cas12a for higher editing efficiency, especially in A/T-rich regions. Enhancements include a streamlined single transcription unit (STU) and temperature-tolerant variants, Mb3Cas12a-R and Mb3Cas12a-RRR, with superior editing performance at lower temperatures. Efficient STU systems in maise and tomato further enable targeted biallelic mutations, expanding Cas12a's application in plant genome research and breeding.
• Optimised CRISPR-Cas9 systems enable targeted editing of Ntab06050 (lignin synthesis) and Ntab0857410 (pectin synthesis) genes in HD and K326 tobacco cultivars using an Agrobacterium-mediated leaf disc method. Eight constructs targeted lignin-related genes, with a 9.4% mutation frequency in NtabCCoAMT, while five constructs focused on pectin-related genes, achieving a 6.2% mutation frequency in NtabGAUT4.
• In a new study, CRISPR-Cas12a proved more effective than Cas9 for miRNA gene knockout in rice, achieving up to 100% editing efficiency and creating larger deletions that disrupt pre-miRNA secondary structures. Targeting nine OsMIRNA genes, Cas12a-generated mutants allowed detailed transcriptome and phenotypic analyses, revealing roles in gene regulation, grain quality, and seed development. This establishes CRISPR-Cas12a as a potent tool for miRNA functional studies in plants.

Disease and stress control

• CRISPR-Cas genome editing in rice has been used to modulate GABA production by targeting the GAD gene and thereby enhance salt tolerance. The engineered gad3-ox1 and gad3-ox2 lines showed increased GABA levels, boosting antioxidant activity, osmotic regulation, and reducing Na+ accumulation. Gene editing significantly improved salt stress resilience compared to wild-type and GABA-deficient lines, offering a pathway for developing salt-resistant rice varieties.
• CRISPR-Cas9-mediated knockout of MIR827 in rice is shown to abolish miR827 production, enhancing resistance to Magnaporthe oryzae infection. While MIR827 overexpression increases blast susceptibility and leaf phosphate (Pi) levels, MIR827 knockout reduces Pi content and strengthens defence gene responses.
• Using CRISPR-Cas9, researchers have edited the EBE_TALE20 region in cassava's MeSWEET10a promoter to disrupt its activation by TALE20_Xam11, a key effector in cassava bacterial blight. The edited cassava plants showed reduced MeSWEET10a expression upon infection, significantly limiting disease symptoms and bacterial growth. This advance offers a promising route to developing blight-resistant cassava without compromising growth or yield.
• CRISPR-Cas9 editing in the elite 'Merlot' grape variety reveals that adding chitosan to callus cell cultures reduces total phenolic compounds while increasing peroxide levels. Using a peroxide-sensing HyPer reporter, researchers identified cells with the MLO7 gene knockout, enhancing pathogen response via an oxidative burst.
• Researchers have identified the GmCG-1 gene, encoding the β-conglycinin α′ subunit, as a target for improving soybean seed quality. Through CRISPR-Cas9 technology, researchers knocked down GmCG-1 and its paralogues GmCG-2 and GmCG-3, producing stable multigene knockdown mutants with improved protein composition and higher salt tolerance.

Agronomic traits

• A novel CRISPR-Cas9 method for boosting gene expression increases protein content in rice and soybean by deleting repressor binding sites in NF-YC4 promoters. Edited rice showed up to 68% more protein in leaves and 17% in seeds, while soybean had up to 25% and 11% increases, respectively.
• CRISPR-Cas9-induced mutations in the TMS5 gene produce tms5 mutants with variable male sterility in rice. These mutants show pollen sterility from 2.05% to 91.1% and seed set rates between 1.14% and 97.36% at restrictive temperatures. This partial fertility suggests incomplete suppression of RNase ZS1, emphasising the need for total RNase ZS1 inactivation in tms5 lines for effective use in hybrid rice breeding.

Industry

• Backed by the Gates Foundation, Pairwise will leverage its Fulcrum™ Platform to apply CRISPR-Cas gene editing in yam through the YOAGE project. By targeting genes responsible for plant architecture, Pairwise aims to develop semi-dwarf yam varieties that suit mechanised farming, reducing labour needs and environmental impact.
• A new market report from The Business Research Company estimates that the CRISPR plants market size will grow from $11.88 billion in 2023 to $23.18 billion in 2028 at a compound annual growth rate (CAGR) of 14.1%. The growth in the forecast period can be attributed to increasing investment in R&D, rising demand for organic and non-GMO products, climate-smart agriculture, increase in plant-based diets, and global water scarcity.

Detection

• Chinese researchers introduce the Cas12aVIP diagnostic method for rapid, sensitive detection of Colletotrichum gloeosporioides complex (CGSC), the primary cause of strawberry anthracnose in China. Targeting the β-tubulin gene, this method integrates recombinase polymerase amplification, CRISPR-Cas12a, and PMNT-ssDNA for visual detection, achieving results in under 50 minutes. Cas12aVIP successfully identifies CGSC in asymptomatic strawberry seedlings, providing a practical, field-ready tool to support healthier strawberry cultivation.
• A new RPA-CRISPR-Cas12a assay enables rapid, sensitive detection of Setophoma terrestris, the causative agent of onion pink root rot. The assay's sensitivity is 0.01 pg/μL - 100 times more sensitive than qPCR. The assay, completed in 40 minutes, combines fluorescence or lateral flow detection and works effectively on infected plant samples, offering a practical tool for quarantine and disease management in agriculture.

Commentaries and opinions

• An article in Forbes describes how CRISPR-Cas and similar gene-editing technologies are transforming agriculture, enabling precise trait improvements in crops, from stress tolerance to yield and nutritional enhancements. Investment is surging, driven by food security challenges, climate change resilience, and sustainability goals. Companies are forming cross-sector partnerships to leverage advanced genetic tools, facilitating traits like pest resistance and reduced environmental impact, while regulatory frameworks evolve to support this innovation globally.

Reviews

• CRISPR/Cas-Mediated Gene Editing in Plant Immunity and Its Potential for the Future Development of Fungal, Oomycete, and Bacterial Pathogen-Resistant Pulse Crops. This review examines recent advances in CRISPR-Cas applications for enhancing disease resistance in pulse crops, highlighting proof-of-concept studies and discussing gene targets potentially useful for broad-spectrum pathogen resistance.
• Geminiviruses and Food Security: A Molecular Genetics Perspective for Sustainable Agriculture in Africa. This review discusses using geminiviruses as vectors for CRISPR-Cas9 and other genome-editing tools to enhance plant resilience in Africa, focusing on manipulating salicylic and jasmonic acid pathways to improve drought resistance and food security.
• Use of CRISPR Technology in Gene Editing for Tolerance to Biotic Factors in Plants: A Systematic Review. This review analyses CRISPR-Cas research on plant resistance to biotic stresses, highlighting key studies, frequently targeted pathogens, and gene-editing enzymes used. It also validates article quality to advance plant breeding strategies for disease resilience.
• Plant conservation in the age of genome editing: opportunities and challenges. This review examines the potential of genome editing to conserve threatened plant species by mitigating genetic vulnerabilities to habitat loss, pathogens, and climate change.
• CRISPR/Cas system-mediated base editing in crops: recent developments and future prospects. This review explores the mechanisms, applications, and future potential of CRISPR-based genome and base-editing systems for precise crop improvement, highlighting DNA and RNA base editors as promising alternatives to traditional gene replacement methods.
• CRISPR/Cas9 mediated genome editing for crop improvement against Abiotic stresses: current trends and prospects. This review examines the use of CRISPR-Cas genome editing to enhance crop resilience to climate-related abiotic stresses, highlighting its potential over traditional transgenic and breeding approaches.