On June 26, 2024, the research team led by Professor Yin Hao from Wuhan University published a research paper titled "Amplification editing enables efficient and precise duplication of DNA from short sequence to megabase and chromosomal scale" in Cell. This study introduces a novel technique, termed Amplification Editing (AE), which facilitates the accurate and efficient replication of genomic sequences, ranging from single gene loci to chromosomal scale with a highly programmable manner

Gene amplification, also referred to as gene duplication, involves the replication of DNA fragments within an organism, resulting in the formation of repeat sequences. The scale of amplification can range from a few base pairs to extensive regions encompassing significant portions of a chromosome. As a structural variation in chromosomes, gene amplification plays a crucial role in evolution, hereditary diseases, and the development and progression of cancer. While existing gene-editing tools enable precise modifications at single loci, they lack the capability for efficient and accurate editing of large-scale genomic structures.

First, the research team validated the method across multiple sites in 11 cell lines. In terms of AE replication length and efficiency, AE can replicate multiple times to form tandem repeats when the replicated DNA is between 20 bp and 8 Kb. When the copy length is 1 Mb, the editing efficiency of AE is up to 73.0%. At a replication length of 100 Mb (close to the average length of chromosomes), the efficiency reaches 3.4%. In terms of the accuracy of AE editing, the next-generation sequencing at junctions and third-generation sequencing data showed that both indels were less than 1%.

Diagram of gene amplification performed by AE

Secondly, AE demonstrated the ability to restore gene expression, amplify miRNAs, and enhance α-globin expression. AE achieved short fragment and Mb-level editing at multiple loci in both human and mouse embryonic stem cells. These findings suggest that AE can accurately construct disease models associated with large-fragment replication.

Finally, the mechanism of AE was preliminarily investigated. In RPE-1 cells with inhibited cell cycles, AE efficiency was significantly reduced. Similarly, in mouse primary neurons, AE was only able to replicate short fragments. These results indicate that Mb-scale AE replication is dependent on cell cycle progression.

The first authors of the paper are Zhang Ruiwen, He Zhou, Shi Yajing, Sun Xiangkun, Chen Xinyu and Wang Guoquan, graduate students of Wuhan University, and Professor Yin Hao is the corresponding author. Wuhan University is the first signatory of the paper.

This work was supported by funding from the National Natural Science Foundation of China and the Key R&D Program of the Ministry of Science and Technology. Additional support was provided by the Instrument and Equipment Sharing Center of the Institute of Medical Sciences at Wuhan University and Zhongnan Hospital of Wuhan University. Yin Hao’s team has long focused on the development and medical application of gene-editing tools, achieving a series of original results.

Full text link:https://doi.org/10.1016/j.cell.2024.05.056