The development of mouse models with precisely targeted integration of DNA mutations are a key technology in experimental cardiovascular research. Advancements of CRISPR-based genome editing technologies facilitate the modification of DNA sequences in the genome. CRISPR-Cas9 induced double strand breaks are repaired by non-homologous end joining (NHEJ), which is the dominant DNA repair mechanism resulting in small insertions and deletions (indels) or by homology-directed repair (HDR) which enable the integration or replacement of defined DNA sequences. However, HDR occurs with low efficiency and precise insertion of longer sequences remains challenging, given that donor DNA templates preferentially multimerizes in the cell by building tandem junctions that integrate in the genome as DNA concatemers.

Methods:
We set out to generate conditional alleles to establish mouse models that allow time-dependent and cell type-specific gene deletions. Therefore, we developed a modified heiCas9 with boosted editing activity (https://www.biorxiv.org/content/10.1101/2021.05.27.445956v2)¹ and used a long donor DNA template containing a loxP-flanked exon that integrates at the target locus after CRISPR-Cas9 mediated DNA double-strand break. To avoid multimerization of donor molecules we 5’ biotinylated DNA donor templates and injected them into mouse embryos. Since donor concatemer integration events cannot be detected by conventional genotyping, we developed a diligent PCR strategy that allow us to distinguish intended single-copy integration (scHDR) from multimeric integration events of donor DNA molecules in the mouse genome.

Results:
Quantitative droplet digital PCR (ddPCR) assays demonstrated that a donor modification by 5’ biotonylation efficiently prevents multimerization in mouse embryos. Thereupon, we found that injecting 5’ biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient and precise single-copy integration of donor DNA. Our dedicated genotyping strategy showed that these precisely modified alleles occurred with a frequency of up to 26%, at different gene loci, indicating that scHDR is dramatically boosted by 5’ biotinylation (https://www.biorxiv.org/content/10.1101/2021.09.30.462539v1)².

Conclusion:
Our fast and precise CRISPR-based genome engineering strategy enables the generation of preclinical genetic mouse models with targeted integration of defined DNA Sequences within 3 months and allows an early characterization of the mutant mice for development of heart diseases. Robust preliminary data can thus be generated within 12 months, whereas this is not possible with conventional gene targeting methods, as the efficiency for precise integrations by these methods is below 1% and the required screening period is very time-consuming.

1.             Thumberger T, Tavhelidse T, Gutierrez-Triana JA, Medert R, Cornean A, Welz B, Freichel M and Wittbrodt J. hei-tag: a highly efficient tag to boost targeted genome editing. bioRxiv. 2021:2021.05.27.445956.

2.             Medert R, Thumberger T, Tavhelidse T, Hub T, Kellner T, Oguchi Y, Dlugosz S, Zimmermann F, Wittbrodt J and Freichel M. Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice. bioRxiv. 2021:2021.09.30.462539.