Figure 2.

Atypical repair outcomes caused by ssODN dimerization in in vitro gene editing. The partial outcome data of one gene editing reaction, carried out using Cas12a in the in vitro system, are shown here. (A) The experimental results. The LacZ gene was repaired with a semi-symmetrical oligo, with one 40 bp and one 29 bp homology arm, with a single base repair (A > C) in the middle. (B) Depiction of the pathway of repair that led to the 48 bp insertions seen in the experimental output. (1) The dimerized ssODN (purple) associated with the cut DNA ends (black) in order to facilitate the generation of the products seen. (2) The dimerized ssODN created a makeshift gap that allowed for DNA synthesis to fill in according to the template (green). Ligation occurs after the green bases are synthesized, incorporating the actual oligo into the top strand of the downstream end as a long 5′-overhang. (3) After ligation, the unincorporated ssODN dissociates from the complex, and the newly-extended 5′-overhang associates with a 5bp stretch of microhomology on the upstream end of the cut site. The 5′-end upstream of this microhomology patch is resected or cleaved, as well as the single mismatched base on the exposed 5′-end of the bottom strand. (4) Ligation occurs after resection on the top strand, leading to a complete top strand. The bottom strand, meanwhile, is filled in via synthesis, using the top strand as a template.