The introduction of small unmarked edits to the genome of insects is essential to study the molecular underpinnings of important biological traits, such as resistance to insecticides and genetic control strategies. Advances in CRISPR genome engineering have made this possible, but prohibitively laborious for most laboratories due to low rates of editing and the lack of a selectable marker. To facilitate the generation and isolation of precise marker-less edits we have developed a two-step method based on CRISPR-mediated cassette exchange (CriMCE) of a marked placeholder for a variant of interest. This strategy can be used to introduce a wider range of potential edits compared with previous approaches while consolidating the workflow. We present proof-of-principle that CriMCE is a powerful tool by engineering three single nucleotide polymorphism variants into the genome of , with 5-41 × higher rates of editing than homology-directed repair or prime editing.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1089/crispr.2022.0026 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Knockout mutation in enhances number of productive tillers in hexaploid wheat.
Front Genome Ed
October 2024
Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Constituent College Pakistan Institute of Engineering and Applied Sciences (PIEAS), Faisalabad, Pakistan.
Article Synopsis
- Recent advances in CRISPR-Cas technology enable precise genetic modifications in crops, particularly for improving wheat traits by targeting specific genes.
- This study focuses on developing knockout mutant lines of an elite wheat cultivar, resulting in a significant increase in both tillers and grains per plant without affecting plant structure.
- The method also utilizes co-transformation techniques to enhance regeneration efficiency of transgenic wheat, demonstrating the potential of CRISPR for boosting grain yield in elite cultivars for future agricultural improvements.
Recent Advances in -Mediated Maize Genetic Transformation Using Immature Embryos and Seedling Leaf Whorl Explants.
Cold Spring Harb Protoc
September 2024
Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA
The introduction of maize genetic transformation in the 1990s brought forth a powerful tool for crop improvement and a deeper understanding of plant genetics. Despite decades of genetics research, however, and the promise of CRISPR-mediated gene editing, maize transformation currently faces several challenges, such as genotype dependence and limitations in explant availability. Indeed, although the most commonly used method, immature embryo transformation, has been improved through optimization of tissue culture media composition and selection methods, the approach is only applicable to a limited number of public genotypes, including B104 and Hi II.
View Article and Find Full Text PDFCHO cell engineering via targeted integration of circular miR-21 decoy using CRISPR/RMCE hybrid system.
Appl Microbiol Biotechnol
August 2024
Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Chinese hamster ovary (CHO) cells, widely acknowledged as the preferred host system for industrial recombinant protein manufacturing, play a crucial role in developing pharmaceuticals, including anticancer therapeutics. Nevertheless, mammalian cell-based biopharmaceutical production methods are still beset by cellular constraints such as limited growth and poor productivity. MicroRNA-21 (miR-21) has a major impact on a variety of malignancies, including glioblastoma multiforme (GBM).
View Article and Find Full Text PDFA P1-like MYB transcription factor boosts biosynthesis and transport of C-glycosylated flavones in duckweed.
Int J Biol Macromol
October 2024
Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Shandong Energy Institute, Qingdao New Energy Shandong Laboratory, Qingdao 266101, China. Electronic address:
C-glycosylated flavones (CGFs) are the main flavonoids in duckweed (Lemna turionifera), known for their diverse pharmacological activities and nutritional values. However, the molecular mechanisms underlying flavonoid metabolism in duckweed remain poorly understood. This study identified a P1-Like R2R3-MYB transcription factor, LtP1L, as a crucial regulator of CGF biosynthesis and transport in L.
View Article and Find Full Text PDFTemporal restriction of Cas9 expression improves CRISPR-mediated deletion efficacy and fidelity.
Mol Ther Nucleic Acids
June 2024
Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Clinical application of CRISPR-Cas9 technology for large deletions of somatic mutations is inefficient, and methods to improve utility suffer from our inability to rapidly assess mono- vs. biallelic deletions. Here we establish a model system for investigating allelic heterogeneity at the single-cell level and identify indel scarring from non-simultaneous nuclease activity at gRNA cut sites as a major barrier to CRISPR-del efficacy both and .
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!