Bridge RNAs direct programmable recombination of target and donor DNA.

Genomic rearrangements, encompassing mutational changes in the genome such as insertions, deletions or inversions, are essential for genetic diversity. These rearrangements are typically orchestrated by enzymes that are involved in fundamental DNA repair processes, such as homologous recombination, or in the transposition of foreign genetic material by viruses and mobile genetic elements1,2. Here we report that IS110 insertion sequences, a family of minimal and autonomous mobile genetic elements, express a structured non-coding RNA that binds specifically to their encoded recombinase. This bridge RNA contains two internal loops encoding nucleotide stretches that base-pair with the target DNA and the donor DNA, which is the IS110 element itself. We demonstrate that the target-binding and donor-binding loops can be independently reprogrammed to direct sequence-specific recombination between two DNA molecules. This modularity enables the insertion of DNA into genomic target sites, as well as programmable DNA excision and inversion. The IS110 bridge recombination system expands the diversity of nucleic-acid-guided systems beyond CRISPR and RNA interference, offering a unified mechanism for the three fundamental DNA rearrangements-insertion, excision and inversion-that are required for genome design.

Transposase-assisted target-site integration for efficient plant genome engineering.

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops1,2. Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3-5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6-9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria10-12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

Detection of EGFR exon 20 insertion mutations in non-small cell lung cancer: implications for consistent nomenclature in precision medicine.

Epidermal growth factor receptor (EGFR) exon 20 insertion mutations (E20ins) are the third most frequent mutations observed in non-small cell lung cancer, accounting for approximately 1-10% of all EGFR mutations. In the era of precision medicine and targeted therapies, consistent naming of genetic alterations is crucial to avoid confusion and errors. However, the annotation of EGFR E20ins mutations has been inconsistent, leading to confusion in the scientific literature and product documentation. In this study, our primary objective was to investigate the usage of different annotation related to EGFR E20ins in independent studies. Additionally, we assessed the distribution of EGFR E20ins mutations and estimated the detection coverage expected from each available EGFR E20ins detection assay. A total of 1,418 EGFR E20ins mutations were collected from six studies (FoundationInsights, Geneseeq Technology Inc, mobocertinib phase I/II trial, poziotinib phase II trial, sunvozertinib phase I trial, and Samsung Medical Center) and reorganised according to Human Genome Variation Society (HGVS) nomenclature. Our analysis revealed that the majority of EGFR E20ins mutations requiring correction were 'insertion' or 'deletion-insertion', which should be appropriately designated as 'duplication'. Additionally, duplicated variants were reported using different annotations in each study, and furthermore, even identical variant sequences were annotated differently within the same study. In all six studies, p.A767_V769dup and p.S768_D770dup were the most frequently observed EGFR E20ins. The Oncomine Dx Target Test showed the highest patient coverage at 77.2%, followed by the Droplex EGFR Mutation Test v2 with a patient coverage of 70.5% for EGFR E20ins patients. To ensure comprehensive coverage in real-world settings, it is essential to standardise the annotations for each variant, for example using the HGVS nomenclature. The accurate classification and analysis of drug responsiveness in EGFR E20ins necessitate consideration of the nomenclature, particularly with respect to the locations where the actual mutations occur.

Novel Alu insertion in the ZEB2 gene causing Mowat-Wilson syndrome.

Alu elements are short, interspersed elements located throughout the genome, playing a role in human diversity, and occasionally causing genetic diseases. Here, we report a novel Alu insertion causing Mowat-Wilson syndrome, a rare neurodevelopmental disorder, in an 8-year-old boy displaying the typical clinical features for Mowat-Wilson syndrome. The variant was not initially detected in genome sequencing data, but through deep phenotyping, which pointed to only one plausible candidate gene, manual inspection of genome sequencing alignment data enabled us to identify a de novo heterozygous Alu insertion in exon 8 of the ZEB2 gene. Nanopore long-read sequencing confirmed the Alu insertion, leading to the formation of a premature stop codon and likely haploinsufficiency of ZEB2. This underscores the importance of deep phenotyping and mobile element insertion analysis in uncovering genetic causes of monogenic disorders as these elements might be overlooked in standard next-generation sequencing protocols.

Alexander disease with a novel GFAP insertion-deletion mutation mimicking progressive supranuclear palsy.

This report presents a case of Alexander disease showing clinical characteristics mimicking progressive supranuclear palsy (PSP). A 67-year-old woman complaining of motor disturbance exhibited severe atrophy of medulla, spinal cord, and midbrain tegmentum, as well as periventricular hyperintensity on cerebral MRI. Genetic analysis identified a novel in-frame deletion/insertion mutation in the exon 3 of the GFAP gene. Interestingly, neurological findings and decreased striatal uptake in dopamine transporter SPECT were suggestive of PSP. A novel GFAP gene mutation found in the present case may cause the unique clinical phenotype, which should be differentiated from PSP.

Expansion and improvement of ChinaMu by MuT-seq and chromosome-level assembly of the Mu-starter genome.

ChinaMu is the largest sequence-indexed Mutator (Mu) transposon insertional library in maize (Zea mays). In this study, we made significant improvements to the size and quality of the ChinaMu library. We developed a new Mu-tag isolation method Mu-Tn5-seq (MuT-seq). Compared to the previous method used by ChinaMu, MuT-seq recovered 1/3 more germinal insertions, while requiring only about 1/14 of the sequencing volume and 1/5 of the experimental time. Using MuT-seq, we identified 113,879 germinal insertions from 3,168 Mu-active F1 families. We also assembled a high-quality genome for the Mu-active line Mu-starter, which harbors the initial active MuDR element and was used as the pollen donor for the mutation population. Using the Mu-starter genome, we recovered 33,662 (15.6%) additional germinal insertions in 3,244 (7.4%) genes in the Mu-starter line. The Mu-starter genome also improved the assignment of 117,689 (54.5%) germinal insertions. The newly upgraded ChinaMu dataset currently contains 215,889 high-quality germinal insertions. These insertions cover 32,224 pan-genes in the Mu-starter and B73Ref5 genomes, including 23,006 (80.4%) core genes shared by the two genomes. As a test model, we investigated Mu insertions in the pentatricopeptide repeat (PPR) superfamily, discovering insertions for 92% (449/487) of PPR genes in ChinaMu, demonstrating the usefulness of ChinaMu as a functional genomics resource for maize.

Common single-base insertions in the VNTR of the carboxyl ester lipase (CEL) gene are benign and also likely to arise somatically in the exocrine pancreas.

The CEL gene encodes carboxyl ester lipase, a pancreatic digestive enzyme. CEL is extremely polymorphic due to a variable number tandem repeat (VNTR) located in the last exon. Single-base deletions within this VNTR cause the inherited disorder MODY8, whereas little is known about VNTR single-base insertions in pancreatic disease. We therefore mapped CEL insertion variants (CEL-INS) in 200 Norwegian patients with pancreatic neoplastic disorders. Twenty-eight samples (14.0%) carried CEL-INS alleles. Most common were insertions in repeat 9 (9.5%), which always associated with a VNTR length of 13 repeats. The combined INS allele frequency (0.078) was similar to that observed in a control material of 416 subjects (0.075). We performed functional testing in HEK293T cells of a set of CEL-INS variants, in which the insertion site varied from the first to the 12th VNTR repeat. Lipase activity showed little difference among the variants. However, CEL-INS variants with insertions occurring in the most proximal repeats led to protein aggregation and endoplasmic reticulum stress, which upregulated the unfolded protein response. Moreover, by using a CEL-INS-specific antibody, we observed patchy signals in pancreatic tissue from humans without any CEL-INS variant in the germline. Similar pancreatic staining was seen in knock-in mice expressing the most common human CEL VNTR with 16 repeats. CEL-INS proteins may therefore be constantly produced from somatic events in the normal pancreatic parenchyma. This observation along with the high population frequency of CEL-INS alleles strongly suggests that these variants are benign, with a possible exception for insertions in VNTR repeats 1-4.

Insertion of YFP at P5CS1 and AFL1 shows the potential, and potential complications, of gene tagging for functional analyses of stress-related proteins.

Crispr/CAS9-enabled homologous recombination to insert a tag in frame with an endogenous gene can circumvent difficulties such as context-dependent promoter activity that complicate analysis of gene expression and protein accumulation patterns. However, there have been few reports examining whether such gene targeting/gene tagging (GT) can alter expression of the target gene. The enzyme encoded by Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) is key for stress-induced proline synthesis and drought resistance, yet its expression pattern and protein localisation have been difficult to assay. We used GT to insert YFP in frame with the 5' or 3' ends of the endogenous P5CS1 and At14a-Like 1 (AFL1) coding regions. Insertion at the 3' end of either gene generated homozygous lines with expression of the gene-YFP fusion indistinguishable from the wild type allele. However, for P5CS1 this occurred only after selfing and advancement to the T5 generation allowed initial homozygous lethality of the insertion to be overcome. Once this was done, the GT-generated P5CS1-YFP plants revealed new information about P5CS1 localisation and tissue-specific expression. In contrast, insertion of YFP at the 5' end of either gene blocked expression. The results demonstrate that GT can be useful for functional analyses of genes that are problematic to properly express by other means but also show that, in some cases, GT can disrupt expression of the target gene.

Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction.

Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries.

Randomly barcoded transposon mutant libraries for gut commensals II: Applying libraries for functional genetics.

The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap. Recent advances in barcoded transposon mutagenesis have dramatically lowered the cost of mutant fitness determination in hundreds of in vitro and in vivo experimental conditions. In an accompanying review, we discuss recent advances and caveats for the construction of pooled and arrayed barcoded transposon mutant libraries in human gut commensals. In this review, we discuss how these libraries can be used across a wide range of applications, the technical aspects involved, and expectations for such screens.

SB Digestor: a tailored driver gene identification tool for dissecting heterogeneous Sleeping Beauty transposon-induced tumors.

Sleeping Beauty (SB) insertional mutagenesis has been widely used for genome-wide functional screening in mouse models of human cancers, however, intertumor heterogeneity can be a major obstacle in identifying common insertion sites (CISs). Although previous algorithms have been successful in defining some CISs, they also miss CISs in certain situations. A major common characteristic of these previous methods is that they do not take tumor heterogeneity into account. However, intertumoral heterogeneity directly influences the sequence read number for different tumor samples and then affects CIS identification. To precisely detect and define cancer driver genes, we developed SB Digestor, a computational algorithm that overcomes biological heterogeneity to identify more potential driver genes. Specifically, we define the relationship between the sequenced read number and putative gene number to deduce the depth cutoff for each tumor, which can reduce tumor complexity and precisely reflect intertumoral heterogeneity. Using this new tool, we re-analyzed our previously published SB-based screening dataset and identified many additional potent drivers involved in Brca1-related tumorigenesis, including Arhgap42, Tcf12, and Fgfr2. SB Digestor not only greatly enhances our ability to identify and prioritize cancer drivers from SB tumors but also substantially deepens our understanding of the intrinsic genetic basis of cancer.

A self-propagating, barcoded transposon system for the dynamic rewiring of genomic networks.

In bacteria, natural transposon mobilization can drive adaptive genomic rearrangements. Here, we build on this capability and develop an inducible, self-propagating transposon platform for continuous genome-wide mutagenesis and the dynamic rewiring of gene networks in bacteria. We first use the platform to study the impact of transposon functionalization on the evolution of parallel Escherichia coli populations toward diverse carbon source utilization and antibiotic resistance phenotypes. We then develop a modular, combinatorial assembly pipeline for the functionalization of transposons with synthetic or endogenous gene regulatory elements (e.g., inducible promoters) as well as DNA barcodes. We compare parallel evolutions across alternating carbon sources and demonstrate the emergence of inducible, multigenic phenotypes and the ease with which barcoded transposons can be tracked longitudinally to identify the causative rewiring of gene networks. This work establishes a synthetic transposon platform that can be used to optimize strains for industrial and therapeutic applications, for example, by rewiring gene networks to improve growth on diverse feedstocks, as well as help address fundamental questions about the dynamic processes that have sculpted extant gene networks.

Deletion of the s2m RNA Structure in the Avian Coronavirus Infectious Bronchitis Virus and Human Astrovirus Results in Sequence Insertions.

Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.

Frequency, underdiagnosis, and heterogeneity of epidermal growth factor receptor exon 20 insertion mutations using real-world genomic datasets.

Epidermal growth factor receptor (EGFR) exon 20 insertion mutations (ex20ins) account for ≤ 12% of all EGFR-mutant nonsmall cell lung cancers. We analysed real-world datasets to determine the frequency of ex20ins variants, and the ability of polymerase chain reaction (PCR) and next-generation sequencing (NGS) to identify them. Three real-world United States NGS databases were used: GENIE, FoundationInsights, and GuardantINFORM. Mutation profiles consistent with in-frame EGFR ex20ins were summarized. GENIE, FoundationInsights, and GuardantINFORM datasets identified 180, 627, and 627 patients with EGFR ex20ins respectively. The most frequent insertion region of exon 20 was the near loop (~ 70%), followed by the far loop (~ 30%) and the helical (~ 3-6%) regions. GENIE, FoundationInsights, and GuardantINFORM datasets identified 41, 102, and 96 unique variants respectively. An analysis of variants projected that ~ 50% of EGFR ex20ins identified by NGS would have been missed by PCR-based assays. Given the breadth of EGFR ex20ins identified in the real-world US datasets, the ability of PCR to identify these mutations is limited. NGS platforms are more appropriate to identify patients likely to benefit from EGFR ex20ins-targeted therapies.

Clinical and Genomic Features of HER2 Exon 20 Insertion Mutations and Characterization of HER2 Expression by Immunohistochemistry in East Asian Non-Small-Cell Lung Cancer.

PURPOSE: HER2-altered non-small-cell lung cancer (NSCLC) represents a diverse subgroup, including mutations, amplifications, and overexpression. However, HER2 exon 20 insertion mutations are emerging as a distinct molecular subtype with expanding therapeutic options. We describe the molecular epidemiology and genomic features of HER2-altered NSCLC in an Asian tertiary cancer center. METHODS: We identified patients with HER2-mutated NSCLC in our institutional database, collating clinicopathological features and treatment outcomes. The genomic landscape of human epidermal growth factor receptor 2 (HER2)-mutated NSCLC was further evaluated using whole-exome sequencing (WES) data from combined local and publicly available data sets. HER2 amplification and overexpression as selection biomarkers in NSCLC were further interrogated using HER2 immunohistochemistry and correlations with WES and RNA sequencing data. RESULTS: Among 1,252 patients with consecutive lung adenocarcinoma undergoing routine next-generation sequencing, the prevalence of HER2 mutations was 3.1%-exon 20 insertion mutations comprised 2.7%. We examined the clinicopathological features in 55 patients with HER2-mutated NSCLC comprising 40 exon 20 insertion and 15 nonexon 20 insertion mutations. The most common exon 20 insertion mutation was HER2Y772_A775dup in 30 (75%), followed by HER2G776delinsVC in five patients (13%). There were limited responses to HER2-directed therapies apart from trastuzumab-deruxtecan, and no responses were seen with immunotherapy monotherapy. Evaluating the genomics features of HER2 exon 20 insertion mutations using WES data revealed low tumor mutational burden (TMB), low incidence of cancer driver comutations, and a predominance of aging mutational signature-similar to EGFR-mutated tumors. In contrast, uncommon (or nonexon 20 insertion) HER2-mutated tumors resembled EGFR wild-type tumors with higher TMB, higher frequency of cancer driver comutations, and greater presence of smoking and APOBEC mutational signature. Finally, in evaluating HER2 immunohistochemistry in all lung adenocarcinoma, there was significant discordance comparing different scoring systems and poor correlation with HER2 RNA expression and HER2 amplification. CONCLUSION: The incidence of HER2 mutations is 3.1% in East Asian nonsquamous NSCLC. HER2 exon 20 insertion-mutated tumors appear genomically distinct from uncommon (nonexon 20 insertion) HER2 mutations, the latter demonstrating higher TMB, co-occurring drivers, and predominant nonaging mutational signature. The therapeutic implications of the genomic and clinical features of HER2-mutated NSCLC warrant further investigation.

Clinical Validation of Companion Diagnostics for the Selection of Patients with Non-Small Cell Lung Cancer Tumors Harboring Epidermal Growth Factor Receptor Exon 20 Insertion Mutations for Treatment with Amivantamab.

Amivantamab, an epidermal growth factor receptor (EGFR)-c-Met bispecific antibody, targets activating/resistance EGFR mutations and MET mutations/amplifications. In the ongoing CHRYSALIS study (ClinicalTrials.gov Identifier: NCT02609776), amivantamab demonstrated antitumor activity in patients with non-small cell lung cancer harboring EGFR exon 20 insertion mutations (ex20ins) that progressed on or after platinum-based chemotherapy, a population in which amivantamab use has been approved by the US Food and Drug Administration. This bridging study clinically validated two novel candidate companion diagnostics (CDx) for use in detecting EGFR ex20ins in plasma and tumor tissue, Guardant360 CDx and Oncomine Dx Target Test (ODxT), respectively. From the 81 patients in the CHRYSALIS efficacy population, 78 plasma and 51 tissue samples were tested. Guardant360 CDx identified 62 positive (16 negative), and ODxT identified 39 positive (3 negative), samples with EGFR ex20ins. Baseline demographic and clinical characteristics were similar between the CHRYSALIS-, Guardant360 CDx-, and ODxT-identified populations. Agreement with local PCR/next-generation sequencing tests used for enrollment into CHRYSALIS demonstrated high adjusted negative (99.6% and 99.9%) and positive (100% for both) predictive values with the Guardant360 CDx and ODxT tests, respectively. Overall response rates were comparable between the CHRYSALIS, Guardant360 CDx, and ODxT populations. Both the plasma- and tissue-based diagnostic tests provided accurate, comprehensive, and complementary approaches to identifying patients with EGFR ex20ins who could benefit from amivantamab therapy.

Transposon insertional mutagenesis of diverse yeast strains suggests coordinated gene essentiality polymorphisms.

Due to epistasis, the same mutation can have drastically different phenotypic consequences in different individuals. This phenomenon is pertinent to precision medicine as well as antimicrobial drug development, but its general characteristics are largely unknown. We approach this question by genome-wide assessment of gene essentiality polymorphism in 16 Saccharomyces cerevisiae strains using transposon insertional mutagenesis. Essentiality polymorphism is observed for 9.8% of genes, most of which have had repeated essentiality switches in evolution. Genes exhibiting essentiality polymorphism lean toward having intermediate numbers of genetic and protein interactions. Gene essentiality changes tend to occur concordantly among components of the same protein complex or metabolic pathway and among a group of over 100 mitochondrial proteins, revealing molecular machines or functional modules as units of gene essentiality variation. Most essential genes tolerate transposon insertions consistently among strains in one or more coding segments, delineating nonessential regions within essential genes.

Molecular Mechanisms in Pentanucleotide Repeat Diseases.

The number of neurodegenerative diseases resulting from repeat expansion has increased extraordinarily in recent years. In several of these pathologies, the repeat can be transcribed in RNA from both DNA strands producing, at least, one toxic RNA repeat that causes neurodegeneration by a complex mechanism. Recently, seven diseases have been found caused by a novel intronic pentanucleotide repeat in distinct genes encoding proteins highly expressed in the cerebellum. These disorders are clinically heterogeneous being characterized by impaired motor function, resulting from ataxia or epilepsy. The role that apparently normal proteins from these mutant genes play in these pathologies is not known. However, recent advances in previously known spinocerebellar ataxias originated by abnormal non-coding pentanucleotide repeats point to a gain of a toxic function by the pathogenic repeat-containing RNA that abnormally forms nuclear foci with RNA-binding proteins. In cells, RNA foci have been shown to be formed by phase separation. Moreover, the field of repeat expansions has lately achieved an extraordinary progress with the discovery that RNA repeats, polyglutamine, and polyalanine proteins are crucial for the formation of nuclear membraneless organelles by phase separation, which is perturbed when they are expanded. This review will cover the amazing advances on repeat diseases.

Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.