Aug 2026· Theriogenology· Vol 266, pp.
118142
· 0 citations· 42 references
Medicine
Abstract
Gene editing in livestock using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) offers a promising approach for genetic improvement in cattle. This study evaluated germline transmission and mutation stability of double-knockout cattle generated by zygote electroporation. Previously reported myostatin/beta-lactoglobulin (MSTN/BLG) and newly generated α-1,3-galactosyltransferase (GGTA1/BLG) double-knockout cattle were produced using CRISPR/Cas9-mediated genome editing. Targeted deep sequencing demonstrated extensive somatic mosaicism across multiple tissues. Computer-assisted sperm analysis (CASA) demonstrated normal sperm motility in MSTN/BLG double-knockout males. Fertilization of wild-type oocytes produced heterozygous embryos, with mutation frequencies of 37.76 ± 10.74% at the MSTN locus and 54.80 ± 7.73% at the BLG locus, as assessed by T7 endonuclease I (T7E1) assay. MSTN/BLG double-knockout sperm were subsequently used for embryo production and for artificial insemination of GGTA1/BLG double-knockout females. Healthy offspring were successfully obtained (n = 3), alongside one stillborn calf. Targeted deep sequencing of all four progenies revealed highly variable allele frequencies that deviated substantially from the approximately 50% expected for heterozygous germline transmission. In contrast, whole-genome sequencing (WGS) results were consistent with Mendelian expectations, underscoring the limitations of PCR-based targeted sequencing for assessing germline transmission in mosaic founders. These results show that CRISPR/Cas9-edited embryos generated by electroporation can develop into healthy, sexually mature cattle capable of germline transmission. While variable transmission rates were observed owing to founder mosaicism, non-mosaic F1 offspring were successfully generated. However, direct, embryo-mediated gene-editing strategies remain technically and economically challenging for large-scale commercial calf production, and reports in cattle are limited. This study provides a reference for future applications of gene-edited embryos and their germline propagation.
The feasibility of generating MSTN/BLG double gene-edited cattle embryos using a single CRISPR/EOCas12i plasmid and SCNT is demonstrated, providing a robust platform for multiplex genome editing aimed at improving meat production and milk traits.
Furui Wang, Lei Chen, Yuting Ning et al.· Frontiers in Genome Editing· 0 citations
Mosaic animals are highly valuable for investigating complex biological processes and cell lineages in vivo. Traditional mosaic techniques in Drosophila, such as the FRT/Flp system, rely on exogenous site-specific recombination sequences, preventing their application to unmodified mutant chromosomes or wild-derived strains. Mosaic analysis by gRNA-induced crossing-over (MAGIC) overcomes this limitation by utilizing the CRISPR/Cas9 system to generate targeted double-strand breaks (DSBs) that induce somatic homologous recombination in precursor cells. Here, we describe a comprehensive protocol for applying MAGIC with a newly developed, genome-wide MAGIC kit. This protocol utilizes optimized gRNA-markers with the Qtg2.1 scaffold for high-efficiency clone induction, alongside improved fluorescent labeling strategies for both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). The procedure details the genetic crossing schemes, temporal induction of clones, and tissue processing for diverse Drosophila cell types. This method enables convenient mosaic analysis across all chromosomes and allows for the study of pericentromeric genes, deficiency chromosomes, and species-specific alleles in interspecific hybrids. Key features • Recombinase-independent: Generates somatic mosaic clones using CRISPR/Cas9 without requiring pre-inserted FRT sequences on the test chromosome. • Genome-wide application: Includes a complete toolkit of pMAGIC and nMAGIC gRNA-markers optimized for all Drosophila chromosomal arms (X, 2L, 2R, 3L, 3R, and 4). • Optimized labeling: Employs destabilized Gal80 for brighter pMAGIC clones and tub-3xHA-BFP/IFP for unambiguous visualization of nMAGIC clones. • Broad compatibility: Applicable to a wide variety of tissues (e.g., neurons, glia, imaginal discs, polyploid tissues) and complex genetic backgrounds, including pericentromeric mutations and deficiencies.
The phenotypic profile of a CRISPR-generated Ent2 mutant line is described and the feasibility of combining genome editing with balancer chromosome strategies in Drosophila is demonstrated, demonstrating the feasibility of combining genome editing with balancer chromosome strategies in Drosophila.
Chikun Li, Lin Li, Ying Chen et al.· Journal of Visualized Experi...· 0 citations
The effects of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated disruption of the myostatin gene (mstnb1) on the growth performance of rainbow trout (Oncorhynchus mykiss) were evaluated. Two specific guide RNAs (gRNAs) targeting the first exon of the target gene were co‐injected into fertilized eggs. While the survival rate of the injected embryos at the swim‐up stage was 23.5%, Sanger sequencing of F0 individuals confirmed the successful generation of mutations. During a 10‐month growing period, quantifiable phenotypic data revealed a significant enhancement in macroscopic somatic growth. The mstnb1‐F0 mosaic fish exhibited an approximate 56% increase in body weight compared to the wild‐type (WT) control group. Specifically, the mutant group reached an average weight of 825.0 ± 12.3 g, significantly outperforming the WT group, which reached 464.1 ± 9.2 g. Our results demonstrate that disruption of the mstnb1 gene using CRISPR/Cas9 technology induces a striking somatic weight gain rather than merely altering microscopic muscle fiber characteristics, demonstrating high biotechnological potential for shortening production time and reducing unit costs in sustainable rainbow trout aquaculture.
A. Bayır, M. Bayır, Wenjing Tao et al.· Journal of the World Aquacul...· 0 citations
A complete CRISPR-Cas9-mediated knockout of the BEL5 gene, encoding a transcription factor, is reported, known as one of the key regulators driving tuber formation, and a regulatory role of BEL5 in the timing of tuber onset but, unexpectedly, its dispensability for tuber development in modern cultivated potato is proposed.
Andrea Zounková, Daniele Chirivì, A. Přibylová et al.· bioRxiv· 0 citations
Background/Objectives: CRISPR/Cas9-mediated genome editing enables efficient generation of knockout mouse models; however, frameshift mutations do not always result in complete loss of function. The factors influencing functional inactivation following frameshift mutations remain incompletely understood. Here, we tested whether frameshift-dominant targeting of exon 1 is sufficient to generate a null allele of Fgf10, a gene essential for limb formation. Methods: Guide RNAs (gRNAs) were selected using a machine learning-based pipeline to favor microhomology-mediated end joining (MMEJ)-dominant repair. Editing efficiency and indel profiles were assessed via amplicon sequencing in mouse embryonic stem cells (mESCs) and preimplantation embryos. Edited embryos were transferred to surrogate females and analyzed at embryonic day 15.5 (E15.5). Results: Amplicon sequencing confirmed >97% editing efficiency and >80% frameshift alleles in both mESCs and preimplantation embryos, with a predominant 7 bp deletion. Despite highly efficient frameshift-dominant editing, most of the E15.5 embryos were morphologically normal, indicating that exon 1 targeting did not reliably produce null phenotypes. In silico analysis suggested the possible presence of alternative downstream translation initiation sites, and our secretion assay supported this possibility. Initiation from a downstream ATG in a +2 reading frame (e.g., 7 bp deletion) may restore the downstream coding sequence and partially preserve protein function. Conclusions: Frameshift mutations in exon 1 of Fgf10 do not consistently result in functional knockout. The functional outcome depends on the specific reading frame and may be influenced by alternative translation initiation and protein domain architecture. These findings highlight important considerations for designing genome editing strategies to achieve complete gene inactivation.
Khanui Lkhagvadorj, Eiichi Okamura, Toshifumi Morimura et al.· Journal of Developmental Bio...· 0 citations
