LbCas12a Nuclease Exhibits Nonspecific 3′→5′ Exonuclease Activity In Vitro

Objective: Despite the widespread use of LbCas12a in genome editing and diagnostic systems, its non-specific activities, particularly the potential to activate collateral activity after introducing a nick in only one strand of the target DNA, or the presence of other nuclease activities such as exonuclease activity, remain poorly characterized. A deeper understanding of these activities is necessary to define the enzyme’s limitations and optimize its application, especially in sensitive diagnostic systems for nucleic acid analysis. Methods: The analysis of non-specific exonuclease activity and the potential for introducing a single-strand nick to activate collateral activity with a guide RNA mutation using the novel TTAА PAM was conducted via fragment analysis. Results and Discussion: Our data demonstrate that LbCas12a exhibits 3′→5′ exonuclease activity in vitro, which is distinct from its canonical collateral cleavage. Furthermore, we found that with the novel TTAA PAM, even in the presence of a single-nucleotide mismatch, the nuclease is unable to cleave either strand of the target DNA. We discuss potential mechanisms for the initiation of this exonuclease activity and its implications for the enzyme’s efficiency in diagnostic applications. Conclusions: The identification of LbCas12a’s exonuclease activity in vitro underscores the importance of considering this factor when developing highly sensitive assays. Mitigating non-specific hydrolysis, potentially through the use of engineered elements like the novel TTAA PAM, could lead to significant improvements in diagnostic performance.