Targeted, efficient sequence insertion and replacement in rice

Rice genes are replaced using chemically modified donor DNA and CRISPR targeting. CRISPR-Cas9 methods have been applied to generate random insertions and deletions, large deletions, targeted insertions or replacements of short sequences, and precise base changes in plants(1-7). However, versatile methods for targeted insertion or replacement of long sequences and genes, which are needed for functional genomics studies and trait improvement in crops, are few and largely depend on the use of selection markers(8-11). Building on methods developed in mammalian cells(12), we used chemically modified donor DNA and CRISPR-Cas9 to insert sequences of up to 2,049 base pairs (bp), including enhancers and promoters, into the rice genome at an efficiency of 25%. We also report a method for gene replacement that relies on homology-directed repair, chemically modified donor DNA and the presence of tandem repeats at target sites, achieving replacement with up to 130-bp sequences at 6.1% efficiency.