Efficient Targeted Transgenesis of Large Donor DNA Into Multiple Mouse Genetic Backgrounds Using Bacteriophage Bxb1 Integrase

Abstract

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene “landing pad” composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies >40% with up to ~43kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development combined with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.

Long Read Sequencing reveals transgene concatemerization and backbone integration following AAV-driven electroporation of CRISPR RNP complexes in mouse zygotes

“…High Molecular Weight (HMW) DNA was obtained using the Monarch  HMW DNA extraction kit for Tissue (T3060L, New England Biolabs), as previously described (Low et al 2022). Briefly, fresh kidney tissues from heterozygous (HET) or Homozygous (HOM) mice were harvested, kept on ice and 20mg of tissue was immediately processed.…”

“…Recently, Oxford Nanopore Technologies (ONT © ) Long Read Sequencing (LRS) has been used in mice to identify the insertion site of randomly integrated transgenes (Bryant et al 2023), and to confirm integration following recombinase-mediated cassette exchange (RMCE) (Low et al 2022). However, to the best of our knowledge, LRS has not yet been used to perform quality control (QC) following AAV-driven gene editing in zygotes.…”