CRISPR-Cas9 Mediates Targeted Gene Replacement and Knockin in Arabidopsis

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′ end and the 3′ 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 methods.

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.

For more information, read the article in Nature Communications.