Insertion of sequences at the original provirus integration site of mouse ROSA26 locus using the CRISPR/Cas9 system

•A Cas9 target that cleaves precisely at the ROSA26 original provirus integration site.•Combining CRISPR/Cas9 with the PITT system can enable insertion of larger DNAs.•Anomalous and mosaic sequence insertions can occur with the CRISPR/Cas9 system.

Targeted transgenic mouse models, where an exogenous gene is inserted into a specified genomic locus to achieve its stable and reliable expression, have been widely used in biomedical research. However, the available methodologies for targeted insertion of sequences require many laborious steps that involve the use of embryonic stem (ES) cells. We recently developed Pronuclear Injection-based Targeted Transgenesis (PITT), a method that uses a recombinase-mediated cassette exchange (RMCE) to enable insertion of sequences at a predetermined genomic locus, such as ROSA26. The PITT technique uses fertilized eggs (instead of ES cells) collected from ‘seed mice’ that contain the RMCE landing pad. The PITT method can rapidly generate reliable targeted transgenic mice; it requires a seed mouse, which in our previous study was generated using ES cell targeting approaches. Here, we demonstrate that seed mice containing the RMCE landing pad can be developed rapidly by using the CRISPR/Cas9 system. One of the CRISPR targets tested in this study enabled the insertion of sequences precisely at the original ROSA26 provirus integration site. We anticipate that using a similar approach, PITT landing pad sequences can be rapidly and precisely inserted at other genomic loci to develop an array of PITT tools. This two-step strategy combines the best features of the two newer technologies—rapid creation of PITT landing pads using the CRISPR/Cas9 system and efficient and precise insertion of larger cassettes at the landing pads using PITT. This study also revealed that anomalous and mosaic sequence insertions can occur with the CRISPR/Cas9 system.

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