The effect of DNA tension on CRISPR/Cas12a cleavage can be reversible.

Abstract

CRISPR/Cas12a (known as Cpf1) is a class 2 type V endonuclease which offers a two-component system for double stranded DNA cleavage. Cas12a is the enzymatic component with an active nuclease domain (RuvC) for cleaving the non-target and target DNA strands in order. CRISPR RNA (crRNA) is the RNA component which is initially processed by Cas12a and later associates with the Cas12a enzyme for the target detection. After Cas12a and crRNA are associated, they form a stable Cas12a/crRNA complex to interrogate the DNA target. Once the complementarity of the guide RNA and the target DNA is maintained, DNA cleavage occurs. Like other CRISPR enzymes, Cas12a is mainly derived from bacteria. However, unlike prokaryotic DNA, eukaryotic DNA faces mechanical stress. For example, DNA stretching and torsional stress can occur during various cellular processes that involve DNA compaction and extraction. As previously reported, the mechanical tension on the DNA reduces or inhibits the CRISPR/Cas12a cleavage reaction. Yet, it remains unknown that if Cas12a cleavage inhibition by tension is reversible. Here I show that the inhibited DNA cleavage can be rescued when the tension is relieved. I employed a high-throughput single-molecule technique to monitor the effect of stretching on hundreds of the target DNA in the same microchamber. The target DNA molecules were tethered to the surface and labeled with magnetic beads. Cas12a/crRNA complexes were introduced when the target DNA was stretched. Later, the target DNA was relaxed by removing the magnetic field. Shortly after this step, I observed the cleavage process of the relaxed DNA tethers. This observation has many implications for the CRISPR/Cas12a gene-editing technique. As it can explain the heterogenous targeting efficiencies reported among the cell cultures with the same guide RNA and targeting strategy.