Predictable NHEJ insertion and assessment of HDR editing strategies in plants

Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indel, its high efficiency mutation is generally not pertinent to precise editing. Homology directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency. Here, we report that single nucleotide insertion at different rice genes is predictable based on DNA sequences at the targeted loci. We also evaluated two distinct strategies to facilitate donor template supply for improving HDR efficiency in Nicotiana benthamiana and rice.