Efficient protein tagging and cis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient NHEJ-mediated targeted insertion method was recently developed by combining CRISPR-Cas9 with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet this approach often led to imprecise insertions with no control over the insertion direction. In this study, we first quantitatively compared the impact of the chemical protection on efficiency of targeted insertion. With the observation that CRISPR- Sp Cas9 could frequently induce staggered cleavages with 5′ 1-nucleotide overhangs, we then evaluated the impact of the donor end structures on the direction and preciseness of targeted insertions. Our study demonstrated that the chemically protected dsODNs with 5′ 1-nt overhangs could significantly improve the precision and direction control of target insertions in all tested CRIPSR targeting sites. Lastly, we applied this method to endogenous gene tagging in Setaria viridis , and cis -regulatory element engineering for disease resistance in rice. Two distinct TAL effector binding elements were directionally inserted into the promoter region of a recessive rice bacterial blight resistance gene at up to 24.4% efficiency. The resulting rice lines with heritable insertions exhibited strong resistance to the infection of Xanthomonas oryzae pv. oryzae pathogen in an inducible and strain-specific manner. ### Competing Interest Statement The authors have declared no competing interest.