Amplification editing: Achieving accurate replication of genome sequences from short fragments to chromosome length

Abstract

Cell recently published the breakthrough of amplification editing (AE) technology by Professor Hao Yin's team. This technology enables the amplification of short sequences to chromosome scale, achieving million-base amplification in both embryonic stem cells and primary cells. This opens up new avenues for precision medicine and genetic research.¹

In the biomedical field, the precise manipulation of the genome has long been a primary goal for scientists. From the debut of CRISPR-Cas9 to the sophisticated application of Prime Editing, each technological iteration represents a profound understanding and clever use of the laws of nature. However, although considerable progress has been made in gene editing technology, particularly in single-gene editing, challenges persist when addressing complex genomic structural variations. The current tools are mainly employed to modify specific gene sites, like point mutations or small insertions/deletions, but encounter numerous difficulties when dealing with large-scale structural variations such as gene amplification. The main issues encompass insufficient precision targeting ability, low efficiency, off-target effects, limited scope of application, and high technical complexity. Given that gene amplification plays a crucial role in genetic diseases and cancer, the inability to effectively identify and correct these variations will restrict the understanding of related disease mechanisms and impede the development of effective treatment strategies. Therefore, the development of precise and effective genome structural variation editing tools is an important direction for future research and treatment of complex genetic diseases.²

Similar to twin prime editing (twinPE),³ AE relies on a pair of prime editing guide RNAs (pegRNAs). The key difference is that in AE, the protospacer-adjacent motifs (PAMs) of the two pegRNAs are positioned on either side of the target site, whereas in twinPE, the PAM is inside the target site. This design allows for the generation of complementary 3′ protruding sequences on both sides of the target site. These sequences form a stable double-stranded DNA structure through annealing and binding, significantly improving sequence amplification and target site editing efficiency. The double-sided PAM design enhances pegRNA binding to the target site, increasing editing success rates. The complementary sequence increases the density of pegRNA binding sites, further improving accuracy and efficiency. Additionally, AE technology retains the complete pegRNA recognition site, making the editing product more stable, reducing synthesis errors, and facilitating subsequent processing and analysis (Figure 1).

The core innovation of AE technology lies in its ability to efficiently and accurately replicate DNA sequences from 20 bp to 100 Mb, while effectively avoiding nonspecific amplification and off-target effects common in traditional gene editing tools. Compared to traditional CRISPR-Cas9, TALENs, and ZFNs technologies, AE technology excels in amplifying large-scale DNA sequences with significant efficiency and accuracy. For example, CRISPR-Cas9 may exhibit off-target effects and low efficiency when amplifying large-scale DNA, TALENs and ZFNs technologies are limited by complex design and insufficient efficiency, and traditional polymerase chain reaction may lack accuracy for long DNA fragments. AE technology overcomes these shortcomings through optimized pegRNA and sgRNA design, offering a more stable and efficient solution. In practical applications, AE technology's efficient sequence amplification and precise regulatory characteristics make it highly effective in stem cell and cell biology research. AE technology can upregulate the expression of noncoding RNA and coding genes, providing new tools for gene expression regulation. It is also suitable for constructing complex chromosome repeat structure models to study the effects of chromosome structural variation on cell function and disease. For instance, in the K562 cell model simulating α-thalassemia, AE technology was used to amplify the HBA1 gene, demonstrating its potential for disease modeling. Importantly, AE technology shows excellent promise in disease treatment. It is expected to correct genomic defects in genetic diseases such as cancer, restoring normal gene function through precise sequence amplification and repair, thus opening new possibilities for genome editing and personalized treatment.

Although AE technology has made significant strides, it still faces key challenges. The editing efficiency of AE is limited by the length of the edited fragment. For instance, while AE achieves a replication efficiency of 73% for 1 Mb fragments, this drops to 1%–3.4% for 100 Mb fragments, with editing results often difficult to detect within 24 h. Additionally, in short fragment repeats, the overlap of pegRNAs and the close proximity of nicks could lead to an increased occurrence of indels. To improve efficiency, future efforts could involve splitting long fragments into smaller ones for step-by-step editing and re-splicing, while carefully designing pegRNA to minimize insertion and deletion risks.

AE efficiency is also affected by the cell cycle. When the cell cycle is inhibited, editing efficiency drops significantly, which limits its application in nondividing cells. This issue is related to the activity of DNA polymerase, which is closely linked to the cell cycle. To address this, introducing appropriate exogenous DNA polymerase could improve efficiency. Future work should focus on optimizing AE tools for different cell cycle stages, exploring cell cycle regulation methods, and understanding AE performance across various cell cycles and nondividing cells. Alternatively, other gene editing technologies could complement AE in finding effective solutions. Controlling repetition frequency is crucial for improving editing accuracy and the reliability of research results, and addressing this will be highly beneficial.

In addition, essentially, AE employs a double nickase method, requiring the recognition of two specific sequences on the target DNA for cutting. Ensuring the specificity and efficiency of editing necessitates the uniqueness and reliability of these sequences in the target genome. However, gene duplication is a prevalent phenomenon in biology. Meanwhile, the sequence characteristics of the genetic locus directly influence the binding efficiency of pegRNA, and diverse cell types can also give rise to variations in the response to pegRNA. Scientists need to establish a comprehensive pegRNA target site screening library and potentially introduce deep learning models to enhance AE technology.

Currently, AE technology is limited to amplifying inherent genomic sequences, and its application to amplifying exogenous sequences or specific mutant sequences remains unexplored. However, two recent studies in Nature demonstrated the bridge RNA system's potential in gene editing, particularly for manipulating large-scale DNA sequences and entire genomes.^{4, 5} This complements AE technology's precision and amplification capabilities in large-scale genome manipulation. The bridge RNA system does not rely on traditional homologous recombination mechanisms but instead uses specific RNA molecules to guide recombinase for accurate DNA double-strand manipulation. It recognizes target and donor DNA through the target-binding loop (TBL) and donor-binding loop (DBL) to achieve precise genome editing. Although currently only applied in Escherichia coli in vitro and in vivo studies, its advantages in gene editing offer new possibilities for AE technology. Specifically, the bridge RNA system could be used to accurately edit the target DNA first, followed by AE technology to efficiently replicate these edited DNA products, achieving precise amplification of the target sequence.

In summary, the advent of AE technology marks a milestone in the field of genome editing. AE provides unprecedented accuracy and breadth for the engineering transformation of genome structures, offering a powerful tool for simulating and treating complex genetic diseases. As the technology matures and its applications expand, AE is poised to lead life sciences into a new era of genome editing.

Jianqiao Shentu analyzed the literature and wrote the manuscript. Jianqiao Shentu and Yitao Zhao drafted the figure. Jianqiao Shentu and Shiwei Duan conceived the idea. Shiwei Duan reviewed and revised the manuscript. All authors gave the final approval of the submitted version. All authors have read and agreed to the published version of the manuscript.

The authors declare no conflict of interest.

The authors have nothing to report.