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Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.

CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome