Genome engineering has emerged as a powerful tool in various scientific and technological fields. In recent years, genome engineering has been successfully used to produce desired traits in various organisms. For example, in the production of biochemicals, genome engineering has been used to produce novel biomolecules and improve the production of essential amino acids, commodity chemicals, and biofuels (Chen et al., 2022; Khan et al., 2014; Tran et al., 2022). In basic research, genome engineering has played a vital role in profiling the relationships between genotypes and phenotypes, which is crucial for understanding gene function and complex regulatory networks (Hilton and Gersbach, 2015). Moreover, genome engineering has significant use in therapeutics. One promising example is gene therapy, in which defective or disease-causing genes are repaired or replaced with functional sequences using genome-editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This technology has been used to correct various mutations that cause cystic fibrosis, sickle cell anemia, and Huntington's disease (Katti et al., 2022; Li et al., 2020). Similarly, genome engineering has shown promising results in cancer research, with CRISPR technology targeting genes and signaling pathways related to cancer growth and metastasis as well as the development of genetically modified T-cells (Katti et al., 2022). Additionally, genome engineering has been used in crop development to produce plants with resistance to pests and diseases, and improved tolerance to environmental stresses (Wally and Punja, 2010; El-Mounadi et al., 2020). These examples illustrate the vast potential of genome engineering in addressing various challenges faced by humanity.

Genome engineering generally involves altering the genetic information of organisms and has a broad range of applications. Therefore, there is a need for a versatile tool that meets various requirements for specific purposes. These requirements include high efficiency, target specificity, and multiplexing power. Additionally, this technology must also be readily available and usable for a wide range of hosts, not just specific species. The integration of large-sized DNA is also an important issue. Genome engineering should be capable of effectively integrating several kilobases (kb) of foreign DNA into the host genome to insert multiple genes or regulatory elements. Continuity is another crucial factor that must be considered. In vivo continuous mutagenesis tools have also gained significant attention owing to the demand for continuous evolution to rapidly search broader sequence spaces.

In response to these demands, various genome engineering technologies have been developed and used. Homology-dependent recombination/recombineering is one of the most prominent examples of genome engineering (Krejci et al., 2012) and uses either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) as templates. In the case of dsDNA, the integration of a fairly large size (up to approximately 12 kb) is feasible (Su et al., 2020). Instead, employing ssDNA allows for high efficiency and multiplexed short size editing, as observed in multiplex automated genome engineering (MAGE) (Wang et al., 2009; Wannier et al., 2021). By utilizing sequence homology, genome-wide targeting of genes, regulatory sequences, and intergenic regions can be achieved. Additionally, desired substitutions, insertions, and deletions can be performed. Another promising application of genome engineering is phage-assisted continuous evolution (PACE) (Esvelt et al., 2011). PACE utilizes the replication ability of a bacteriophage and combines it with the function of a target protein, allowing simultaneous mutagenesis and selection of the target protein in the cytoplasm of Escherichia coli. PACE has been successfully used to engineer a wide range of proteins, including protein toxins, proteases, and gene-editing enzymes such as Cas9 and base editors (Miller et al., 2020). This technology has enabled the development of mutant proteins with improved functions compared to wild-type proteins or even completely new functions. The CRISPR/Cas system is currently considered the most significant genome-editing tool available (Knott and Doudna, 2018). CRISPR/Cas is a genome editing technique that uses the bacterial immune system to accurately find and alter DNA sequences in living cells. It uses guide RNA to direct Cas enzymes to a specific location in the genome, causing a double-stranded break. The natural DNA repair mechanisms of cells can then repair the break using either non-homologous end-joining or homology-directed repair, resulting in targeted gene editing. Multiple genome engineering technologies are currently under development to accomplish specific objectives through the fusion of different enzymes with mutated Cas proteins. Several of these advancements have been reported in the literature. These previous works include the development of a base editor that introduces point mutations into the target sequence by incorporating base deaminase (Gaudelli et al., 2017), an EvolvR system that induces diverse mutations using error-prone polymerase (Halperin et al., 2018), and a prime editor that enables precise editing of the desired sequence through reverse transcriptase (Anzalone et al., 2019).

However, the aforementioned gene editing technologies face some challenges. One existing method relies on specific homologous recombination systems that can only be used in a small number of bacterial species due to the lack of effective phage-derived proteins. The functionality of phage-derived proteins is restricted to certain bacterial species, and their efficiency significantly decreases when attempting to insert large DNA fragments via recombination. Unintended insertions or deletions (indels) may occur because of double-strand breaks (DSB) in the CRISPR/Cas9 system. Moreover, the homology-directed repair method mainly works in actively dividing cells; therefore, the majority of cell types are excluded from current gene replacement/correction procedures. The occurrence of DSBs is not necessary in the context of CRISPR-derived editors, such as base and prime editors, although the editing capacity is restricted to a small range of several tens of base pairs. Techniques for site-specific integration of DNA sequences larger than a kilobase in bacteria are hampered by low efficiency, the need for multiple vectors, and the difficulty of multiplexing.

Mobile genetic elements (MGEs) are DNA segments that can move within or between genomes. DNA transposons, retrotransposons, retrons, and CRISPR-associated transposons (CASTs) are few examples of MGEs (Fig. 1). MGE-based genome engineering technologies have been successfully used to overcome various limitations of the existing genome engineering methods. First, MGE has a broad host range; therefore, it can be applied to various organisms without the need for host-specific factors. This versatility allows the widespread use of MGE-based techniques in many prokaryotic and eukaryotic cells. Second, MGE-based genome engineering enables genome-wide mutagenesis, which can induce genetic changes across the entire genome of an organism. This capability is valuable for studying gene function, identifying targets for therapeutic interventions, and exploring the relationships between genotypes and phenotypes. For this objective, CRISPR is widely recognized as a tool for genetic screening across entire genome. However, achieving this task involves the deployment of individual single guide RNAs (sgRNAs) designed to target all genes constituting the genome. This can be achieved through the utilization of oligonucleotide pools or a plasmid library. In contrast, DNA transposons, classified as MGE, exhibit the ability of random integration, providing the benefit of easily obtaining a comprehensive collection of genome-wide insertional mutants. Another important advantage is their capacity to efficiently integrate large DNA fragments into the host genome, particularly when the DNA exceeds a size of 10 kilobases. These capabilities facilitate the insertion of multiple genes or regulatory elements, enabling the modification of complex genetic systems, thus opening up possibilities for creating synthetic pathways, engineering novel traits, or optimizing biological processes. Moreover, MGE-based genome engineering enables simultaneous targeting of multiple loci using guide RNA. While not all genome engineering technologies support multiplexing, CRISPR achieves this through multiple sgRNA expression. However, the efficiency of simultaneous editing with CRISPR is hindered by its double-strand break mechanism. Base editors and prime editors, which utilize the Cas9 without inducing double-strand breaks, also face limitations in multiplex editing. This is due to the inability of the Cas9 used in these systems to process gRNAs from a single array, necessitating complex expression constructs and causing delivery challenges. Conversely, Cas6 protein in most CAST systems allows the cleavage of the guide RNA array and their maturation to individual guide RNAs, exhibiting notable efficiency in multiplexing. Finally, MGE-based genome engineering enables in situ ssDNA generation. This is particularly useful for applications such as homology-directed repair, in which the presence of ssDNA is crucial for promoting specific DNA repair mechanisms. In summary, MGE-based genome engineering offers a broad host range (DNA transposon, retron, CAST), the ability to perform genome-wide mutagenesis (DNA transposon), continuous mutagenesis in defined cargo region (retrotransposon), efficient integration of large DNA sequences (DNA transposon, CAST), multiplexing capabilities (retron, DNA transposon), and in situ ssDNA generation (retron) (Fig. 1). These advantages make MGE-based approaches highly versatile and powerful tools for gene editing and genome engineering.

In this review, we introduce genome engineering tools developed using MGEs and their respective applications (Table 1, Table 2). We focused specifically on the detailed components, mechanisms of action, and features of each tool according to the type of MGE used. Furthermore, we provide an overview of the wide range of cellular applications of MGE-based genome engineering methods. The perspectives and challenges of these tools are discussed in detail to gain a better understanding of the current and future prospects of genome engineering technologies based on MGEs.