DNA large fragment deleting by compact, sequence-motif-free and specific TaqTth-hpRNA assisted with the microhomology-mediated end joining pathway

<p><span>A DNA editing tool TaqTth-hpRNA was developed in this study, composed of a compact recombinant TaqTth nuclease (832 aa) and a simple hairpin-RNA guiding probe (hpRNA).&nbsp;<em>In vitro</em> biochemical studies showed the TaqTth-hpRNA efficiently cleaves artificially synthesized ssDNA without stringent sequence motif like PAM. It can also cleave the genomic DNA of <em>E. coli</em> with ~80% efficiency.&nbsp;The TaqTth-hpRNA cleavage of genomic DNA in mammalian cells generated products with large fragment deletions mediated by the microhomology-mediated end joining (MMEJ) pathway.&nbsp;In addition, the cleavage was sensitive to mismatches in targeted regions, which was applied to specific damage of the <em>APP^lon</em> mutation in Alzheimer&rsquo;s disease without disrupting the <em>APP^wt</em> locus. It is worth mentioning that the <em>APP^lon</em> sequence has only one base difference from that of <em>APP^wt</em>. The characteristics of small size, no PAM requirement, high specificity, and large deletion products make the TaqTth-hpRNA a potential therapeutic strategy for treating autosomal dominant disorders in the future.</span></p>