Transgenic animals are of vital importance for dissecting complex biological interactions as well as for modelling human diseases and testing therapeutic interventions. Gene targeting using embryonic stem cells has produced a large number of successful mouse models for basic and translational research. However, the technique itself remains a labour-intensive and time-consuming process. By enabling direct modifications of the embryonic genome, the establishment of CRISPR-Cas9 has immensely facilitated the generation of transgenic mouse lines and eliminated prior limitations imposed by the use of embryonic stem cells.

Thanks to recent advances, small insertions and deletions can readily be produced through electroporation of CRISPR factors into zygotes. Yet, not all loci are equally amenable to genetic modifications. Furthermore, insertion of longer fragments or other large-scale modifications remain challenging. Based on the data obtained over several years of performing CRISPR-mediated genetic modifications, we found that the epigenetic landscape could provide indications on the integration efficiency to be expected [1]. This information will help us while choosing the insertion strategy and can further help to improve integration efficiencies.

An additional concern is that multiple tandem integrations of the provided repair template at the target locus are more frequently observed than expected [2, 3]. Depending on the size and type of integration, these concatemeric insertions can be challenging to detect using standard PCR and/or restriction enzyme based screening methods. Therefore, we are currently working on establishing and optimizing a long-read sequencing pipeline to efficiently and cost-effectively screen and validate genetic modifications. This will allow us to assure that only animals with the desired modifications are entering breeding programs and experiments.

1. Baur, A., et al. Can We Use Historical Data to Predict Optimal Targeting Strategies for Efficient Knock-in Generation of Mouse Models ? presented at ISTT 2023. Houston TX.
2. Codner, G.F., et al., Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biol, 2018. 16(1): p. 70.
3. Skryabin, B.V., et al., Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events. Science Advances, 2020. 6(7): p. eaax2941.