Despite recent progress in plant genome editing, site-directed chromosomal integration of exogenous DNA is still of low efficiency. One approach to mitigate this problem is increasing the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carry their recognition motifs. When fused to a CRISPR-associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis using chimeric proteins between LbCas12a as a CRISPR-associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean. Two fusion protein configurations were tested to integrate a 70nt oligonucleotide donor into a commercially important target site using ex planta and in planta transformation. Site-directed integration rates of the donor DNA, when tethered to the fusion protein reached about 20% in plants and were up to three-fold higher than in untethered controls. Integrations via either homologous recombination or non-homologous end joining were promoted by tethering in similar fashion. This study is the first demonstration of HUH EN associated tethering to improve site-directed DNA integration in plants.

Covalent tethering of exogenous DNA to LbCas12a promotes its site-directed integration into the soy genome

ACS Fall 2021

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