Mechanism of Permissive Cleavage Activity of TevCas12a
Abstract
We created a novel dual endonuclease called TevCas12a. TevCas12a is a fusion of the I-TevI nuclease and linker domains to Cas12a. The goal was to create a dual endonuclease that made precise double-strand breaks leaving non-compatible ends for exogenous DNA insertion and specific deletions. TevCas12a was expected to make two double-strand breaks, one by I-TevI and the other from Cas12a. However, when tested in vitro, TevCas12a cut the DNA substrate multiple times, rapidly degrading the substrate. We call this activity permissive cleavage. TevCas12a activity is guide RNA dependent, but not guide RNA sequence specific, it can degrade any DNA substrate with a non-targeted guide RNA. Sequencing of the TevCas12a cleavage products with Oxford Nanopore revealed that most of the non-guide directed cuts were within I-TevI’s cleavage motif, indicating that I-TevI is responsible for the cuts. A mechanism is proposed for how I-TevI and Cas12a work together to permissively cleave DNA.