Breakthrough in Gene-Editing Technology Made by Chinese Researchers

A breakthrough in gene-editing technology has been made by Chinese researchers who have developed a new system to improve prime editing efficiency in certain crops. This innovation, known as SmartPE, aims to overcome one of the major limitations of prime editing: its relatively low efficiency in dicot plants such as tomatoes and soybeans.

The key to SmartPE lies in introducing mismatches into the reverse transcriptase templates of two prime editing guide RNAs (pegRNAs). By combining this strategy with a cis-regulatory element and an engineered reverse transcriptase, researchers hope to boost the precision and speed of gene editing. This could pave the way for more efficient crop modification.

The study behind SmartPE was led by Professor Gao Caixia from the Institute of Genetics and Developmental Biology (IGDB) at the Chinese Academy of Sciences (CAS). The research has been published in a leading scientific journal, providing valuable insights into the potential applications of this new technology. The findings have significant implications for agriculture and crop improvement.

In dicot plants, prime editing currently struggles to achieve high efficiency levels, with off-target events and byproducts often occurring. This makes it difficult to introduce precise genetic changes into these crops. By addressing this limitation, SmartPE could revolutionize the way researchers approach crop modification and potentially lead to more sustainable agricultural practices.

To test the effectiveness of SmartPE, researchers first inserted a cis-regulatory element called START between the promoter and the Cas9 coding sequence in Arabidopsis plants. This resulted in a significant increase in editor expression, with transcript levels rising by 2.1-fold. Consequently, editing efficiency at four target sites was also boosted by around 1.8-fold.

The team behind the new system, SmartPE, sought to further improve its performance by introducing complementary mismatched bases into the RTTs of both pegRNAs.

This modification led to a significant increase in average editing activity at the four targets, with efficiency rising more than sixfold and reaching approximately 10%. The enhanced system was dubbed SmartPE, indicating its smart approach to prime editing.

The researchers investigated how the arrangement of mismatches within the pegRNAs would impact editing efficiency. They found that continuous mismatches or those spaced 3 bp apart yielded the highest levels of efficiency, with some targets achieving up to 20% editing activity.

In contrast, intervals of 6 bp or more between mismatches resulted in a sharp decline in efficiency. This pattern diverged from what was observed in single-pegRNA systems, highlighting the unique characteristics of SmartPE.

The placement of mismatches within the pegRNAs also seemed to play a crucial role in editing efficiency, with those at both ends of the editing window contributing more significantly than those in the middle.

The efficiency of prime editing has been significantly improved for several dicot plants using a new system developed by scientists.

SmartPE, the newly created system, produced higher efficiencies in generating single amino-acid substitutions in soybean and tomato targets, with rates 1.6 to 11.1 times greater than previous methods.

In experiments involving stable transformation, SmartPE showed promising results in tomato hairy roots, achieving homozygous and heterozygous edits at the SlOR2 locus at rates of 7.3% and 12.5%, respectively. In contrast, standard prime editing only yielded heterozygous edits and chimeras.

The development of SmartPE provides a practical framework for applying prime editors in dicot crops, including Arabidopsis, tomato, soybean, and rapeseed, based on empirical design principles that consider mismatch density and distribution.