Amplification editing empowers in situ large-scale DNA duplication

Main text

Repetitive sequences in genomes play crucial biological roles involved in maintaining chromosome structure or the development of diseases. Modeling naturally occurring large-scale repetitive sequences will facilitate the exploration of their functions and elucidate their underlying mechanisms. However, constructing programmable large-scale repetitive sequences on chromosomes remains a challenge. The Yin laboratory’s latest study published in Cell reported a significant breakthrough in the generation of large-scale repetitive sequences on chromosomes, with the method demonstrating feasibility across various human- and mouse-derived cell lines, including diploid, haploid, primary, and embryonic stem cells (ESC) (Figure 1).¹ The researchers ingeniously altered the recognition region of nCas9 from a PAM-in orientation (where the PAM sequences are within the target region) to a PAM-out orientation (where the PAM sequences are at the end of the target region), resulting in in situ tandem duplication of chromosomal segments. Notably, they achieved 100 Mb replication at the chromosomal scale for the first time. Furthermore, amplification editing (AE) can generate multiple rounds of replication, producing tandem repeat sequences that range from 20 bp to 8 kb with fewer insertion-deletion mutations (indels).

Figure 1.

Schematic of amplification editing for chromosomal duplication

Several methods for constructing repetitive sequences have been developed; these methods are commonly based on Gibson assembly or enzymatic ligation in vitro. However, these methods are largely restricted by the size of the DNA, especially up to the hundred-kb level, as large sizes affect the integrity of extraction and delivery. AE technology enables duplications to be created directly in vivo, bypassing the problem of delivering repetitive sequences and achieving duplications from the nucleotide level to the chromosome level. In addition, other methods have been employed via the introduction of specific seed sequences (short repetitive unit sequences) that carry selection markers and leveraging selection pressure to generate random copies of the seed sequences. For example, in the assembly of synthetic Saccharomyces cerevisiae chromosome 12, Dai et al. integrated rDNA seed sequences containing a hyg1 mutation to form Mb-level rDNA clusters by increasing the concentration of hygromycin B during propagation, thus avoiding the direct assembly of repetitive rDNA sequences.² In contrast, AE is based on CRISPR-Cas technology, which can theoretically be programmed to directly duplicate hundred-Mb chromosomal segments at arbitrary genome sites. Hence, compared with other approaches, AE enables the construction of large-scale DNA tandem repeats without selection pressure.

In addition, the study by Yin et al. highlighted how AE can be applied in fundamental research and disease modeling. AE can be used to precisely overexpress coding genes, amplify noncoding regions, and upregulate microRNA expression. For example, in the myeloid leukemia cell line K562 with a single HBA1 copy, AE increased the number of copies of the HBA gene and successfully increased the α/γ-bead protein ratio. In addition, disease-associated regions can be replicated in embryonic stem cells via AE, which demonstrates that AE serves as a powerful tool for chromosome manipulation and disease modeling.

Despite important breakthroughs in AE technology, there are still some challenges in constructing large-scale repetitive sequences. First, large-scale repetitive sequences may become destabilized during propagation, especially in cells with high homologous recombination capacity. Our study revealed that the assembly of Mb-sized immunoglobulin heavy chain (IGH) sequences containing repetitive sequences was unstable in Saccharomyces cerevisiae. Therefore, ensuring that the assembled large-scale DNA containing repetitive sequences remains stable is still an urgent problem. Second, AE mainly produces in situ tandem repeats for preexisting sequences in the genome, which makes it difficult to amplify exogenous or synthetic sequences. AE technology in combination with synthetic genomics techniques, such as the incorporation of large-fragment DNA assembly and delivery technology^3,4 or genome integration technology,⁵ may broaden its application, allowing for the de novo design and assembly of synthetic DNA sequences and their delivery and integration into the targeted genome. In this way, AE technology can achieve customized large-scale synthetic DNA replication.

In summary, excellent advances have been made in the development of AE technology, which can efficiently and accurately generate chromosomal duplications in situ. Moreover, the versatility of AE in multiple cell types and the feasibility of its use in ESC are substantially valuable in the construction of cellular and animal disease models. Since AE relies on CRISPR-Cas technology, which is widely available in bacteria, fungi, and cells, AE has the potential to be employed in more organisms.

Acknowledgments

This study was supported by the National Key R&D Program of China (2019YFA0903800 and 2021YFC2102500), the National Natural Science Foundation of China (32471483), and the Natural Science Foundation of Tianjin (23JCYBJC00220).

Declaration of interests

The authors declare no competing interests.

Published Online: October 16, 2024