Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase
Year: 2022
Type: article
Abstract: Efficient, targeted integration of large DNA constructs represent a significant hurdle in genetic engineering for the development of mouse models of human disease and synthetic biology research. To address this, we developed a system for efficient and precise, targeted single-copy integration of large transgenes directly into the zygote using multiple mouse genetic backgrounds. Conventional approaches, such as random transgenesis, CRISPR/Cas9-mediated homology-directed repair (HDR), lentivirus-based insertion, or DNA transposases all have significant limitations. Our strategy uses in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) to efficiently generate precise single-copy integrations of transgenes. This is achieved using a transgene “landing pad” composed of dual heterologous Bxb1 attachment (att) sites in cis , pre-positioned in the Gt(ROSA)26Sor safe harbor locus. Successful RMCE is achieved in att carrier zygotes using donor DNA carrying cognate attachment sites flanking the desired donor transgene microinjected along with Bxb1-integrase mRNA. This approach routinely achieves perfect vector-free integration of donor constructs at efficiencies as high as 43% and has generated transgenic animals containing inserts up to ∼43kb. Furthermore, when coupled with a nanopore-based Cas9-targeted sequencing (nCATS) approach, complete verification of the precise insertion sequence can be achieved. As a proof-of-concept we describe the creation and characterization of C57BL/6J and NSG Krt18-ACE2 transgenic mouse models for SARS-CoV2 research with verified heterozygous N1 animals available for experimental use in ∼4 months. In addition, we created a diverse series of mouse backgrounds carrying a single att site version of the landing pad allele in C57BL/6J, NSG, B6(Cg)-Tyrc-2J/J, FVB/NJ, PWK/PhJ, 129S1/SvImJ, A/J, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, and DBA/2J for rapid transgene insertion. Combined, this system enables predictable, rapid creation of precisely targeted transgenic animals across multiple genetic backgrounds, simplifying characterization, speeding expansion and use.
Source: Scientific Reports
Institution: Jackson Laboratory
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,190
Awards: The Jackson Laboratory Gene Editing Testing Center (JAX-GETC), Development and Validation of a Novel Cas13a and Nanoparticle Guide-RNA Delivery System that Allows Precise Ablation of Host Macrophage Populations in a Humanized Mouse Model, Resources for Comparative Mendelian Disease Genomics, Cancer Center Support (CORE) Grant
OpenAlex ID: W4225737911