CRISPR /Cas9‐mediated mstnb1 disruption enhances growth in rainbow trout

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/Cas12a-mediated allele engineering of SmAPRR2 and SmGLK2 reveals complementary control of fruit peel and flesh chlorophyll pigmentation in eggplant

A multiplex CRISPR/Cas12a system is established in eggplant accession MEL3, representing, to the authors' knowledge, the first application of this nuclease for genome editing in eggplant and demonstrating the potential of Cas12a for functional genomics, allele engineering, and precision breeding in eggplant.

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 .

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

In this study, a CRISPR/Cas9-based genome-editing approach was used to introduce mutations in the equilibrative nucleoside transporter 2 (Ent2) gene in Drosophila melanogaster. Guide RNAs targeting the coding region of Ent2 were designed and co-injected with Cas9 mRNA into w1118 embryos. Mutant alleles were identified by Sanger sequencing and maintained as a stable Ent2*/CyO heterozygous line using a balancer chromosome. Subsequently, we evaluated body weight, climbing ability, survival rate, and the activities of superoxide dismutase (SOD) and catalase (CAT) in fruit flies at 22 °C and 25 °C, respectively. The results indicate that at both 22 °C and 25 °C, the body length and weight of Ent2*/CyO fruit flies were significantly reduced compared to the w1118, and their development was delayed. At 22 °C, the overall lifespan of Ent2*/CyO flies was slightly longer than that of the w1118, whereas at 25 °C, no significant difference was observed. Regarding locomotor ability, the climbing performance of heterozygous flies was significantly lower than that of the w1118 at both temperatures, with males being more severely affected. In addition, the antioxidant enzyme activities of CAT and SOD in Ent2*/CyO fruit flies were significantly reduced, indicating a clear impairment of antioxidant capacity. These results describe the phenotypic profile of a CRISPR-generated Ent2 mutant line and demonstrate the feasibility of combining genome editing with balancer chromosome strategies in Drosophila. This study provides a methodological framework and a genetic resource for future investigations of genes associated with metabolism and environmental responses.

Ef fi cient epigenetic editing of the zebra fi sh slc45a2 gene using the CRISPR/dCas9 system and tandem guide RNAs

Precise dual-gene knockout of MSTN and SOCS2 via cytidine base editing enhances muscling and growth in goats