A team of Chinese researchers led by Professor GAO Caixia from the Institute of Genetics and Developmental Biology at the Chinese Academy of Sciences has unveiled groundbreaking advancements in genome editing. Their work, published in the journal Cell on August 4, introduces two innovative technologies collectively known as Programmable Chromosome Engineering (PCE) systems. These systems achieve precise DNA manipulations ranging from kilobase to megabase scale, particularly in higher organisms such as plants.
The announcement comes as a significant leap forward in genetic engineering, addressing long-standing limitations of the widely used Cre-Lox recombinase system. Despite its potential, Cre-Lox has faced challenges due to reversible recombination reactions, the complexity of engineering its tetrameric structure, and the presence of residual Lox sites post-recombination. These issues have historically limited its broader application in precise chromosomal manipulation.
Overcoming Historical Challenges
The research team tackled these challenges head-on, developing novel methods to enhance the technology’s precision and efficiency. First, they constructed a high-throughput platform to modify recombination sites rapidly, proposing an asymmetric Lox site design. This innovation led to the creation of new Lox variants that significantly reduce reversible recombination activity, achieving a reduction by over tenfold compared to previous models.
Meanwhile, the team advanced their AiCE (AI-informed Constraints for protein Engineering) model, a system that integrates structural and evolutionary constraints with inverse folding models. This led to AiCErec, a recombinase engineering method that optimizes Cre’s multimerization interface, resulting in a variant with a recombination efficiency 3.5 times that of the wild-type Cre.
Lastly, the researchers developed a scarless editing strategy using prime editors. By designing Re-pegRNA, they enabled re-prime editing on residual Lox sites, effectively replacing them with the original genomic sequence and ensuring seamless genome modifications.
Innovative Platforms and Applications
The integration of these innovations has culminated in the creation of two programmable platforms: PCE and RePCE. These platforms allow for flexible programming of insertion positions and orientations of Lox sites, facilitating precise, scarless manipulation of DNA fragments from kilobase to megabase scale in both plant and animal cells.
Key achievements include targeted integration of large DNA fragments up to 18.8 kilobases, complete replacement of 5-kilobase DNA sequences, chromosomal inversions spanning 12 megabases, chromosomal deletions of 4 megabases, and whole-chromosome translocations.
As a proof of concept, the researchers successfully used this technology to create herbicide-resistant rice germplasm with a 315-kilobase precise inversion, demonstrating its transformative potential for genetic engineering and crop improvement.
Implications for the Future of Genetic Engineering
This pioneering work not only addresses the historical limitations of the Cre-Lox system but also opens new avenues for precise genome engineering across various organisms. According to experts, the implications of these advancements are far-reaching, potentially revolutionizing fields such as agriculture, medicine, and biotechnology.
Dr. Li Wei, a genetic engineering expert not involved in the study, commented,
“This breakthrough represents a significant step toward realizing the full potential of genome editing technologies. The ability to perform precise, large-scale DNA manipulations with high efficiency and accuracy could lead to unprecedented advancements in crop resilience and medical therapies.”
Looking forward, the research team plans to explore further applications of their technology in different organisms and environments, aiming to refine the systems for broader use. As the field of genetic engineering continues to evolve, the PCE systems developed by Professor GAO’s team are poised to play a pivotal role in shaping its future.
