A novel needleless delivery of Cas9 ribonucleoprotein complex in Bemisia tabaci embryos for gene editing

Tiny but powerful: protoplast isolation in Solanum melongena L. and RNPs mediated genome editing

Traditional genetic transformation approaches relying on Agrobacterium tumefaciens for the delivery of CRISPR/Cas9 reagents usually provide plants that stably integrate the gene construct in their genome. To meet the EU commission’s proposal for a new legislation on plants obtained by new genomic techniques (NGTs), it is important to develop new protocols that produce transgene-free genome edited plants (NGT category 1). Protoplasts are a promising platform, since delivery of CRISPR/Cas9 reagents as ribonucleoproteins (RNPs) is effective in cells lacking their wall. This allows genetic modifications from a transient application, leaving no traces in the recipient genome apart from the desired targeted mutations. With the aim of implementing transgene-free editing of eggplant (Solanum melongena L.), we adapted and improved a protocol previously established in potato and tomato for the isolation of protoplasts from cotyledonary leaves and subsequent CRISPR/Cas9 reagents delivery. Isolated protoplasts were subjected to in vitro culture and regeneration, and the first shoots were regenerated from calli approximately 4–5 months after isolation. Alongside, two transfection protocols were tested for the delivery of RNPs into eggplant protoplasts, one using polyethylene glycol (PEG) in two concentrations (25% and 40%) and one exploiting two formulations of lipofectamines (Lipofectamine CRISPRMAX™ and Lipofectamine™ 3000), all targeting SmChl_H gene, whose inactivation can cause a chlorotic phenotype. Efficient callus regeneration from transfected protoplasts was obtained and the editing efficiency (calculated as the percentage of edited calli on the total of calli that underwent sequencing) was evaluated. 25% PEG treatment provided the highest editing efficiency, and fully edited biallelic calli were retrieved, showing the expected chlorotic phenotype. Even if the efficiency of in vitro regeneration of plants from calli still needs improvement, edited plants were regenerated from protoplasts, representing the first report of RNP mediated genome editing in eggplant protoplasts.

A Reproducible Electroporation Strategy for CRISPR-Cas9 RNP and mRNA Delivery in Fish Embryos

Cpf1(Cas12a)-based genome editing in the filamentous cyanobacterium Nostoc punctiforme

The filamentous cyanobacterium Nostoc punctiforme is a key model organism used to study several aspects of cyanobacterial biology, including development, nitrogen-fixing symbioses with plants, and secondary metabolites, among others. While N. punctiforme is amenable to genetic manipulation, traditional approaches for the generation of mutant strains using homologous recombination are slow, requiring prolonged outgrowth under antibiotic selection to ensure isogenic mutant populations. CRISPR-based genome editing using Cpf1 (Cas12a) was recently shown to be an effective means of rapid generation of isogenic mutants in several cyanobacteria. In this study, Cpf1-based genome editing tools were developed for N. punctiforme. A total of 19 unmarked, in-frame deletion mutants were successfully constructed using Cpf1-targeted cleavage along with homology directed repair (HDR). The length of the homology arms (HAs) on the homologous repair template (HRT) used for HDR was found to be a critical factor for successful deletion of target genes, with some requiring up to 4 kb HAs to acquire mutant exconjugants. A strategy for allelic replacement was also developed by introducing an exogenous target site in place of the deleted genes, which could subsequently be targeted for cleavage and repaired with an HRT containing altered alleles of the genes of interest. Additionally, a single-step cloning strategy was devised, allowing for rapid assembly of editing plasmids, and improved conjugation protocols for genetic transfer from E. coli to N. punctiforme were implemented. Collectively, these tools and protocols should enhance the pace and ease of conducting genetic studies in this important model cyanobacterium.

In vitro CRISPR/Cas9–RNP cleavage of CcYUC1 in Coffea canephora

Efficient Endogenous Tagging in the Sea Urchin, Lytechinus pictus, Using CRISPR/Cas9-mediated Split-Fluorescent Protein Knock-In

Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K1-10). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG211, the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (∼130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.

Field-Deployable RPA-CRISPR/Cas12a Detection of Decapod Iridescent Virus 1 in Shrimp.

Decapod iridovirus 1 (DIV1) is a highly lethal pathogen that infects decapod crustaceans including Litopenaeus vannamei, causing mass mortality in cultured shrimp and severe economic losses worldwide. The ATPase gene is a highly conserved region within the DIV1 genome, plays a critical role in viral replication and represents an ideal target for molecular diagnostic development. In this study, we established a rapid, sensitive and field-adaptable detection platform for DIV1 by integrating recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system. RPA enables efficient isothermal amplification of target nucleic acids, achieving exponential enrichment of the target nucleic acids and exerting the function of signal amplification. While the CRISPR/Cas12a system upon crRNA-guided specific recognition of the amplicon, triggers robust trans-cleavage activity against reporter probes for signal generation and readout. After systematic optimization, the RPA reaction was performed at 38°C for 10 min and the CRISPR-Cas12a reaction was conducted at 37°C for 20 min. The integrated two-step workflow completed detection within 40 min, with a limit of detection of 2.3 × 101 copies/μL. Specificity evaluation confirmed that the RPA-CRISPR/Cas12a assay exclusively recognised DIV1 without cross-reaction with other major shrimp pathogens. Further validation using clinical shrimp samples demonstrated stable and reliable performance, supporting its practical utility in aquaculture settings. In conclusion, the established CRISPR/Cas12a-based detection platform provides a robust technical tool for early warning and on-site rapid screening of DIV1, facilitating timely disease control and risk management in shrimp farming.