Homology-directed gene repair (HDR), in which the gene of interest is
knocked in to the genome or replaced to an undesired DNA sequence, is a
powerful tool in CRISPR-Cas9-mediated genome editing. However, this
technique has limited successes in higher plants, although it has been
applied to other organisms such as yeast, mice, and fruit fly.
Scientists from Shanghai Center for Plant Stress Biology and Center for
Excellence in Molecular Plant Sciences developed a "sequential
transformation" gene-targeting strategy to target endogenous DNA glycosylase
genes ROS1 and DME in Arabidopsis. They first tested the applicability of
the method by knocking in ROS1-GFP gene and found that the strategy can
result to a stably inherited genomic locus as large as 1.6 kb.
To further test their method, they knocked in GFP at the 5$B!l(B end and the 3$B!l(B
end of DME and found that the gene was stably transferred. They tested amino
acid substitution within a conserved region of DME and found that the
substitution was stable and heritable. They also investigated the status of
methylation in the edited regions and found unaffected DNA methylation of
the targeted genomic locus. These results were easily determined using PCR
However, researchers concluded that further study must be done to better
understand and improve gene targeting through HDR, so that it can be applied
to other plants, especially crops.