CRISPR/Cas9-Mediated Generation and Characterization of an Ent2*/CyO Drosophila melanogaster Strain.

CRISPR/Cas9-Mediated Editing of Bsr-d1 and Pi21 Enhances Blast Resistance in a High-Quality Rice Maintainer Line

Rice (Oryza sativa L.) is a staple food crop worldwide, and improving disease resistance is a core target in rice breeding. In this study, we employed CRISPR/Cas9 genome editing to modify the coding sequence (CDS) of two susceptibility genes, Bsr-d1 and Pi21, in the elite maintainer line Gengxiang B to enhance its blast resistance. We generated Bsr-d1/Pi21 double homozygous mutants via Agrobacterium-mediated genetic transformation. Quantitative RT-PCR revealed significantly suppressed transcript accumulation of both target genes in the edited lines compared with the wild type Gengxiang B. Upon inoculation with Magnaporthe oryzae, multiple defense-related marker genes were markedly upregulated in the double mutants. Phenotypic assays demonstrated significantly reduced disease severity for both leaf and panicle blast in the edited lines compared with the wild type. Importantly, no statistically detectable differences were found between the double mutants and wild-type plants for key agronomic or grain quality traits. Collectively, these results demonstrate that CRISPR/Cas9-mediated editing of susceptibility loci generates genetically stable blast-resistant rice germplasm without compromising agronomic traits or grain quality, providing valuable genetic resources for future rice varietal improvement.

Establishment of a CRISPR/Cas9-mediated system for targeted editing of the MFS gene in mint

A substantial decrease in menthofuran content in the essential oil of the edited line #10 compared to the wild-type control is revealed, thereby demonstrating a viable strategy for improving mint essential oil quality through genome-editing.

Programmable RNA targeting with clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas7-11 in zebrafish embryos and mammalian cells

Findings establish Cas7-11 as a precise and efficient RNA knockdown tool for functional studies in embryonic development and stem cell biology, providing a versatile alternative to DNA-based gene-editing approaches.

CRISPR/Cas9-mediated generation of a homozygous MT4 knockout mouse embryonic stem cell line.

Metallothionein 4 (MT4) is a low-molecular-weight, cysteine-rich metal-binding protein belonging to the metallothionein family. It exhibits unique skin developmental and differentiation inhibitory activity when functionally impaired and regulates skin cell growth and disease through multiple mechanisms. However, its exact role in cell fate determination remains unknown. Here, we utilized the CRISPR/Cas9 system to generate a homozygous Mt4 knockout (Mt4-/-) mouse embryonic stem cell (mESC) line. This cell line maintains normal morphology, pluripotency, and the ability to differentiate into all three germ layers. It provides a valuable resource for investigating the mechanisms underlying skin diseases caused by MT4 gene mutations.

Targeted Gene Expression Modulation Using CRISPR/dCas9 to Investigate Pathogenic Outcomes in Tomato.

Homologous knock-in processes facilitated by CRISPR-Cas9 in Aurantiochytrium and disrupting the gene for DNA ligase IV prevents transformation events

This study explores advanced molecular breeding techniques for Aurantiochytrium limacinum , a eukaryotic microorganism valued for its industrial production of DHA and astaxanthin. We first employed the CRISPR-Cas9 system to simultaneously disrupt two genes: crtIBY , a multifunctional carotenoid synthesis gene used as a visual marker, and lig4 , which is involved in non-homologous end joining (NHEJ). This genetic disruption caused the wild-type orange colonies to turn into a white colony (Δ lig4 -Δ crtIBY -TA#6), indicating the loss of carotenoid production. Subsequently, we successfully demonstrated marker recycling by repairing the crtIBY gene using single-strand oligodeoxynucleotide (ssODN) templates, which restored the orange phenotype (Δ lig4 -TB#18). Further investigation into the Δ lig4 strain (TB#18) revealed that its overall transformation efficiency dropped significantly compared to that of the wild-type strain when using only a bleomycin resistance expression cassette with two crtIBY homologous regions. However, when we combined the bleomycin resistance expression cassette with CRISPR-Cas9 ribonucleoproteins (RNPs) during electroporation, the Δ lig4 strains (TA#6 and TB#18) exhibited higher homologous recombination-type efficiencies through double-crossover-type events than the wild-type. These results indicate that while Lig4 is important for general transformation against zeocin, disrupting the NHEJ pathway enhances precise gene targeting. This study demonstrated the potential of these CRISPR-Cas9 systems to serve as a robust foundation for the functional genomics and metabolic engineering of A. limacinum .