Profiling sequence preference of I-TevI chimeric gene editors by de-coupling DNA cleavage from DNA targeting

Profiling the cleavage preferences of gene editing nucleases is crucial for selecting robust target sites andfor understanding tolerance to target site nucleotide substitutions. Here, we develop an in vitro approachusing Oxford Nanopore sequencing to map the cleavage preferences of I-TevI-based chimeric gene editors. By manipulating in vitro salt concentrations, we found that DNA cleavage by the I-TevI (Tev) nuclease domain at CNNNG sites was de-coupled from the targeted site. For TevCas12a, this non-targeted cleavage activity mapped to Tev CNNNG cleavage motifs optimally positioned within a 30-bp window up- or down-stream of a Cas12a TTTV PAM site. Non-targeted cleavage did not require Cas12a nuclease activity or specific Cas12a gRNA targeting. Similar non-targeted products were observed in low salt buffer conditions for TevSaCas9, Tev-meganuclease and Tev-zinc finger editors. Cas12a and SaCas9 tolerance to target-site mismatches was also sensitive to buffer salt concentration. Oxford Nanopore sequencing revealed a remarkably similar Tev CNNNG cleavage preference at different salt concentrations and in different fusion contexts. More generally, our work highlights the sensitivity of gene editors to in vitro reaction conditions and how these conditions can be leveraged to functionally dissect the activity of individual domains of chimeric gene editors.This entry contains FASTQ files of all Oxford Nanopore sequencing samples referred to in this manuscript.