In a study published online in Nature Biotechnology on July 6, a team led by Dr. ZHU Jiankang from Shanghai Center for Plant Stress Biology, Center for Excellence in Molecular Plant Sciences of the Chinese Academy of Sciences reported important progress in plant genome editing. Using modified DNA fragments as donors, they established an efficient sequence insertion and replacement technology in rice. The high efficiency of this gene targeting method would greatly facilitate plant genetic research and breeding. 

In recent years, CRISPR/CAS-mediated genome editing technology has played an important role in crop research and breeding. However, CRISPR/CAS-mediated gene knockout technology can only generate random insertions and deletions at targeted sites. The efficiency of sequence insertion and replacement has been very low, which greatly limits its application in plant research and breeding. Therefore, there is an urgent need to establish a more efficient system of sequence insertion and replacement. 

Plant genome editing is a key research field in Dr. ZHU’s lab. As a major problem in this field, efficient sequence insertion and replacement has been an important research goal for the laboratory in recent years. After the successful establishment of virus-mediated homologous recombination (HDR) and various base editing techniques, they began to play with donor DNA fragments to overcome the shortcomings of existing methods.  

Nucleic acid modification has been widely used in human RNAi therapy, nucleic acid vaccine and other medical fields. The researchers in this study found that simultaneous phosphorothioate-linkage modification and phosphorylation modification of a donor fragment could greatly enhance its insertion efficiency.  

They have successfully targeted various regulatory elements to 14 genomic loci, including translation enhancers, transcriptional regulatory elements, and even the entire promoters. The analysis of 1393 T0 rice plants showed that the insertion frequencies of this method could be up to 47.3% and the average efficiency was 25%.  

The researchers also achieved simultaneous insertion at four loci. It is expected that the establishment of this technology will make targeted insertion a routine experiment like CRISPR/Cas9-mediated gene knockout, which will be widely used in plant research and breeding. 

Building on the efficient insertion method, the researchers designed an elegant strategy for sequence replacement, which is called the tandem repeat-HDR strategy (TR-HDR). The conventional HDR frequency is extremely low, but in their earlier experiments, they found that the frequency of HDR between tandem repeats in the genome is very high. They utilized this phenomenon to design TR-HDR to achieve sequence replacement by inserting the modified donor into the target site. Using this technology, the researchers successfully achieved precise sequence replacements and protein tag fusions at five gene sites, with frequencies up to 11.4%. 

The technological breakthrough in this study will be very helpful to plant research and greatly facilitate crop improvement.