The human gut microbiome possesses varied impacts on human health and disease, influencing obesity (Van Hul and Cani, 2023), non-alcoholic fatty liver disease (Safari and Gerard, 2019), type 2 diabetes (Zhu and Goodarzi, 2020), inflammatory bowel disease (Vijay and Valdes, 2022), and cancer (Schmidt et al., 2018). The emergence of omics technologies has transformed our structural and functional grasp of the human gut microbiota and expanded our understanding of the linkages between human health and gut microbiota. Moreover, advances in the culturing of human gut microbes (Lagier et al., 2018; Matar and Bilen, 2022), have been critical for mechanistic studies. Utilizing isolated human gut microbes alongside gnotobiotic animals, several investigations have demonstrated a causal relationship between specific species or strains present in the human gut microbiome and diseases (Boleij et al., 2015; Routy et al., 2018).

Genetic manipulation tools for human gut commensal bacteria are essential to disentangle the responsible molecular mechanisms. Currently, various genome editing tools, including CRISPR/Cas systems, homologous recombination, transposon-based systems, and integrase-based systems, have been successfully utilized in a few cultured gut commensal bacteria (Table 1). However, most gut commensal bacteria lack efficient genome editing tools. A critical obstacle for genome editing of gut commensal microbes is the successful administration of DNA vectors to the target bacteria. This is essential and is impacted by DNA delivery methods, available genetic elements (including promoter, ribosome-binding site (RBS), and terminator), and bacterial defense systems.

Another genome editing tool generation approach includes species- and site-specific genome editing within the complex gut microbial community (Rubin et al., 2022; Gencay et al., 2023). This approach has the potential to deepen our understanding of the intricate relationships between hosts and gut commensal microbes, particularly for uncultured gut bacteria. In addition, in situ genome editing can be utilized for valuable living biotherapeutics to engineer gut commensals (Nethery et al., 2022). However, an essential prerequisite for this approach is the efficient and precise delivery of genome editing cargo to gut commensal bacteria throughout the complicated and vast microbial community of the host.

Currently, functional examinations of gut commensal microbes are still in their infancy. Compared to the development of single-gene editing methods, progress in the development of high-throughput genome editing methods for rapid functional exploration of multi-gene networks, or entire genomes, has lagged. High-throughput mutation library construction techniques, including transposon insertion sequencing (TIS) and CRISPR interference (CRISPRi) libraries, have been applied in a small number of model gut commensal bacteria for functional genomic research (Cain et al., 2020). Moreover, targeted high-throughput editing methods such as MAGE (Multiplex Automated Genome Engineering) and SAGE (Serine Recombinase-Assisted Genome Engineering) have been implemented for some model bacteria strains (Wannier et al., 2021). However, exploration of multiple-gene correlations remains a challenge in the study of gut commensal bacteria (Arnold et al., 2016), which hinders a complete understanding of host-microbe interactions. This underscores the pressing need to create high-throughput genomic tools for manipulating various gut microbes.

In recent years, rapid development in genome editing tools for human gut commensal bacteria has been observed. This review aims to deliver a comprehensive summary of general strategies used for genome editing gut commensal bacteria. Moreover, it highlights the difficulties associated with cargo DNA delivery for genome editing into gut commensal bacteria, both in vitro and in situ, and proposes possible optimizations. The review also investigates the challenges and prospects of high-throughput genetic manipulation of gut commensal bacteria.

This article is composed of three parts. Part 1 discusses the progress and difficulties of in vitro editing of gut bacteria, particularly in delivering cargo DNA into target gut bacteria. Part 2 discusses strategies and difficulties for in situ editing of gut bacteria in model animals, including bacterial conjugative delivery and phage delivery. Part 3 highlights methods for high-throughput gene editing within gut bacteria, including a transposon mutagenesis library, CRISPRi mutant library, and multiplex automated genome engineering (MAGE).

The development of genetic tools for gut commensal bacteria encompasses several key steps (Fig. 1A). The bacterial strain of interest must be isolated and characterized via the collection of fecal samples, culturing of the bacteria, and biochemical and genetic tests, including genome sequencing must also be conducted. Once the strain is identified, the following step is to develop an approach for adequate delivery of the genetic editing system into the bacterial cells. Gene expression elements, cargo DNA delivery, and bacterial defense systems impact this process. Ultimately, upon successful delivery and integration of the genetic cargo, genome editing of the target bacteria can be carried out, enabling specific genomic changes or modifications to be made. These steps are required for developing effective genetic tools for gut commensal bacteria, allowing deeper understanding and manipulation of their characteristics and functions.

The human gut microbiome encompasses over 5000 bacterial species, with the human microbiome containing over 10 million non-redundant genes (Thursby and Juge, 2017; Tierney et al., 2019). Disentangling the functional aspects of these genes requires successful delivery of genome-editing reagents into non-model gut commensal bacteria. For instance, Jin et al. systematically characterized a pipeline for cargo DNA transfer methodology, successfully achieved genome editing in 88 isolates from human gut microbes, and performed a functional examination of the microbial gene baiH within the host's microbiome (Jin et al., 2022). Gene delivery efficiency to gut microbes is impacted by various factors, including delivery methods, available genetic elements, and bacterial defenses (Fig. 1B-1D). Comprehending and optimizing these factors are necessary for achieving effective gene delivery and manipulation throughout gut commensal bacteria.