Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals

Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (alpha LA-LPH Tg goat, Akr1A1(eGFP/eGFP) Tg mouse, and NF kappa B-Luc Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (alpha LA-AP1 GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5 ' homologous arm inverted repeat donor plasmid (5 ' IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, p < 0.05). However, the KI rate of the 3 ' inverted terminal repeat/inverted repeat donor plasmid (3 ' ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3 '-overhang integration into the host genome via homologous recombination machinery.

Automated summary

This study found that flexible cruciform DNA structures play an important role in the integration of exogenous DNA into the chromosomes of transgenic animals. These DNA structures act as legible templates, promoting the integration of DNA into linear DNA or double-strand break alleles. Experimental results using the CRISPR/Cas9 system in NIH3T3 cells showed that the knock-in rate was significantly higher when using a 5' homologous arm inverted repeat donor plasmid that matched with ROSA gRNA compared to a knock-in reporter donor plasmid.