Hi! While performing an in vitro Cas9 RNP cleavage assay on PCR products to test my RNP efficiencies, I noticed some of the sgRNAs result in 3 or more cleavage products instead of two as expected.

On the attached image you can see what I expect on the left: one small band indicating the small cleavage products for the 3' and 5' of the DNA fragment and two-ish larger ones above. Whereas on the right, above the smallest band at the expected size (~600 bp), a larger one appears at ~1000 bp. This band does not correspond to the secondary cleavage product of the large fragment which should be ~3200 bp.

To me this seems to indicate that the RNP cleaves two sites within the DNA fragment but this seems highly unlikely.

After doing some research I wasn't able to find any resource explaining this phenomenon. I am hoping someone can help me here.

Thanks in advance for the answers.