The Impact of DNA-DNA Base Pairing on CRISPR-Cas12a Target Specificity

CRISPR-Cas12a is a highly specific Cas endonuclease characterized by reversibility during R-loop formation that allows Cas12a to more effectively discriminate against mismatches compared to Cas9. However, the role of DNA-DNA base pairing in Cas12a’s target specificity is not fully understood. CRISPR-Cas12a target specificity occurs primarily when contacts are formed reversibly. We hypothesize that the ability of DNA to reform DNA-DNA base pairs at the expense of RNA-DNA base pairs increases reversibility and therefore specificity. In the present work, we eliminate DNA-DNA base pairs, predicting that R-loop formation would be faster and less specific. To test this, single DNA mismatches were generated throughout R-loop positions in both a traditional duplex target and a “bubble” target that lacks Watson-Crick base pairs in the R-loop region. Cis-cleavage and binding data indicate that R-loop formation occurs more rapidly in the absence of DNA-DNA base pairing compared to in the duplexed target. Results also indicate that at certain positions, mismatch specificity is decreased when DNA-DNA base pairs are absent from the target sequence. A model is suggested where target specificity is reduced in the absence of DNA-DNA base pairing throughout the entirety of R-loop positions.