Background: Chromosomal deletions and inversions are important genetic events driving cancer progression. However, conventional methods for detecting DNA rearrangements require laborious indirect assays. To facilitate mechanistic studies of CRISPR-mediated DNA rearrangements, we developed fluorescent reporters to rapidly quantify deletion and inversion events between two sgRNA-guided Cas9 cleavage sites.

Results: Using these reporters, we identified that inversion depends on the NHEJ enzyme LIG4. We also observed that CRISPR/Cas9 mediates both deletions and inversions, which we validated for a 50kb Pten genomic region in adult mouse liver in vivo. Moreover, we discovered diverse yet sequence-specific small insertional or deletional mutations (indel) at the rearrangement fusion sites by deep sequencing. In contrast to the prevailing notion that Cas9 generates blunt DNA breaks at 3nt upstream of PAM, we detected Cas9 cleavage at 4th nt on the non-complementary strand, leading to staggered DNA breaks.

Conclusions: The direct and flexible properties of these CRISPR reporters provide a fast and quantifiable method to investigate mechanisms of chromosomal rearrangements. The surprising feature of staggered Cas9 cleavage is consistent with the pattern of CRISPR-mediated indels in mammalian cells.