Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice

Journal article

Abstract Background The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route. Results We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods. Conclusions Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9–4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction

Authors: Duddy, G; Courtis, K; Horwood, J; Olsen, J; Horsler, H

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: BioMed Central
• Journal: BMC Biology
• Volume: 22
• Issue: 1
• Pages: 26-26
• Article number: 26
• Publication date: 2024-02-02
• DOI: 10.1186/s12915-024-01834-z
• EISSN: 1741-7007
• ISSN: 1741-7007
• Language: English
• Keywords: Genetics; Electroporation; Transduction (biophysics); Gene knockout; Blastocyst; Embryonic stem cell; Stem cell; Zygote; Cas9; Gene knockin; CRISPR; Recombinant DNA; Cell biology; Embryogenesis; Somatic cell nuclear transfer; Microinjection; Gene delivery; Genetic enhancement; Adeno-associated virus; Gene targeting; Biology; Gene; Germline; Vector (molecular biology); Genome editing