Cas9‐Rep fusion tethers donor <scp>DNA</scp><i>in vivo</i> and boosts the efficiency of <scp>HDR</scp>‐mediated genome editing

Genome editing based on the homology-directed repair (HDR) pathway enables scar-free and precise genetic manipulations. However, the low frequency of HDR hinders its application in plant genome editing. In this study, we engineered the fusion of Cas9 and a viral replication protein (Rep) as a molecular bridge to tether donor DNA in vivo, which enhances the efficiency of targeted gene insertion via the HDR pathway. This Rep-bridged knock-in (RBKI) method combines the advantages of rolling cycle replication of viral replicons and in vivo enrichment of donor DNA at the target site for HDR. Chromatin immunoprecipitation indicated that the Cas9-Rep fusion protein bound up to 66-fold more donor DNA than Cas9 did. We exemplified the RBKI method by inserting small- to middle-sized tags (33-519 bp) into 3 rice genes. Compared to Cas9, Cas9-Rep fusion increased the KI frequencies by 4-7.6-fold, and up to 72.2% of stable rice transformants carried in-frame knock-in events in the T₀ generation. Whole-genome sequencing of 6 plants segregated from heterozygous KI lines indicated that the knock-in events were faithfully inherited by the progenies with neither off-target editing nor random insertions of the donor DNA fragment. Further analysis suggested that the RBKI method reduced the number of byproducts from nonhomologous end joining; however, HDR-mediated knock-in tended to accompany microhomology-mediated end joining events. Together, these findings show that the in vivo tethering of donor DNAs with Cas9-Rep is an effective strategy to increase the frequency of HDR-mediated genome editing.