Molecular heterogeneity and treatment outcome of EGFR exon 20 insertion mutations in Chinese patients with advanced non-small cell lung cancer: insights from a large-scale real-world study.

INTRODUCTION: This retrospective study aimed to investigate treatment patterns and outcomes in patients with NSCLC harboring EGFR20ins in China. EGFR20ins mutations are associated with poor responses to EGFR-TKIs, and limited real-world data exist regarding the efficacy of various treatment modalities. METHODS: In this retrospective, single-center study, treatment outcomes, including PFS and ORR, were evaluated for different treatment regimens based on imaging assessments. The impact of mutation heterogeneity on treatment efficacy was also explored. RESULTS: Data from 302 patients diagnosed with NSCLC with EGFR20ins were analyzed. EGFR-TKI monotherapy demonstrated suboptimal PFS compared to platinum-based chemotherapy in the first-line setting (3.00 m vs. 6.83 m, HR = 3.674, 95%CI = 2.48-5.44, p < 0.001). Platinum plus pemetrexed plus bevacizumab combination therapy showed improved PFS and ORR compared to platinum plus pemetrexed (7.50m vs. 5.43 m, HR = 0.593, 95%CI = 0.383-0.918, p = 0.019). In later-line treatments, monotherapy with EGFR-TKIs or ICIs exhibited suboptimal efficacy. The specific EGFR20ins subtype, A763_Y764insFQEA, showed favorable responses to EGFR-TKIs in real-world settings. CONCLUSIONS: This large-scale real-world study provides valuable insights into the treatment patterns and outcomes of NSCLC patients with EGFR20ins mutations in China. These findings contribute to the understanding of EGFR20ins treatment and provide real-world benchmark for future clinical trials and drug development.

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum.

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Current clinical practice and physicians' insights on Chinese patients with advanced non-small cell lung cancer habouring epidermal growth factor receptor 20 insertion mutation.

BACKGROUND: The present study aimed to investigate physicians' perspectives on the diagnosis and treatment decisions for patients with non-small cell lung cancer (NSCLC) harbouring epidermal growth factor receptor (EGFR) exon 20 insertion (exon20ins) mutations in a real-world setting in China using an online questionnaire. METHODS: This study was performed via the CAPTRA-Lung collaboration between December 9, 2022 and March 6, 2023. The questionnaire was distributed digitally to physicians around China and was comprised of three sections: basic characteristics of surveyed physicians, diagnosis and treatment status of NSCLC patients with the EGFR exon20ins-mutation, and physicians' perspectives on treatment options. Physicians who treat more than 10 patients with advanced NSCLC every month and who have treated patients with advanced EGFR exon20ins-mutant NSCLC in the past six months were involved in this study. RESULTS: A total of 53,729 questionnaires were distributed and 390 valid ones were collected. The EGFR mutation test was performed in 80.9% and 59.9% of patients receiving first-line or second-line therapy and beyond (hereinafter "second-line")therapy, respectively. In terms of treatment options, chemotherapy plus antiangiogenic therapy was the most common treatment option (30.0% of patients in first-line settings; 25.0% of patients in second-line settings), and a certain proportion of patients received novel EGFR exon20ins-targeted agents (including tyrosine kinase inhibitors [TKIs] and bispecific antibodies) in first- or second-line settings, which accounted for 11.9% and 15.7% of all treated patients, respectively. Additionally, physicians reported the highest satisfaction score for the efficacy and safety of targeted agents. Most physicians believed that EGFR exon20ins-targeted TKIs represented the most promising treatment option (80.2% in first-line treatment and 73.3% in second-line treatment). Among several novel agents under study, sunvozertinib has received the highest recognition for efficacy and safety. CONCLUSIONS: This study investigated the current diagnosis and treatment status and physicians' perspective, of patients with EGFR exon20ins-mutant NSCLC. The results highlight significant unmet clinical needs in this subgroup of patients. EGFR exon20ins-targeted TKIs were recognized as the most promising treatment regimen and may benefit more patients considering their awareness and acceptance of targeted therapy.

Screening of pathogenicity-deficient Penicillium italicum mutants established by Agrobacterium tumefaciens-mediated transformation.

Blue mold, caused by Penicillium italicum, is one of the main postharvest diseases of citrus fruits during storage and marketing. The pathogenic mechanism remains largely unclear. To explore the potential pathogenesis-related genes of this pathogen, a T-DNA insertion library of P. italicum PI5 was established via Agrobacterium tumefaciens-mediated transformation (ATMT). The system yielded 200-250 transformants per million conidia, and the transformants were genetically stable after five generations of successive subcultures on hygromycin-free media. 2700 transformants were obtained to generate a T-DNA insertion library of P. italicum. Only a few of the 200 randomly selected mutants exhibited significantly weakened virulence on citrus fruits, with two mutants displaying attenuated sporulation. The T-DNA in the two mutants existed as a single copy. Moreover, the mutant genes PiBla (PITC_048370) and PiFTF1 (PITC_077280) identified may be involved in conidia production by regulating expressions of the key regulatory components for conidiogenesis. These results demonstrated that the ATMT system is useful to obtain mutants of P. italicum for further investigation of the molecular mechanisms of pathogenicity and the obtained two pathogenesis-related genes might be novel loci associated with pathogenesis and conidia production.

[Chinese Expert Consensus on the Standardized Diagnosis and Treatment of 
Non­small Cell Lung Cancer with EGFR Exon 20 Insertion Mutations (2024 Edition)].

The standard clinical practice of managing the non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutations was elaborated in Chinese expert consensus on non­small cell lung cancer with EGFR exon 20 insertion mutations (2023 edition), and this rare subset has gradually attracted attention recently. With the deepening of treatment area exploration and the approval of new targeted drugs, there are more options for the diagnosis and treatment of EGFR ex20ins positive NSCLC patients. Therefore, based on the previous version of consensus, the expert panel has updated this consensus on the standardized clinical diagnosis and treatment of EGFR ex20ins mutation NSCLC through reference to literature and clinical data, and combined with the experts' own clinical experience. The updated recommendations includes disease congnition, testing methods, therapy and recent relevant clinical trials for NSCLC patients with EGFR ex20ins mutation, in order to provide better medication reference for clinical physicians.
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A case study of Duchenne muscular dystrophy caused by Alu element insertion in DMD gene and analysis of its gray-hair symptoms.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive genetic disorder caused by mutations in the DMD gene, which leads to a deficiency of the dystrophin protein. The main mutation types of this gene include exon deletions and duplications, point mutations, and insertions. These mutations disrupt the normal expression of dystrophin, ultimately leading to the disease. In this study, we reported a case of DMD caused by an insertion mutation in exon 59 (E59) of the DMD gene. The affected child exhibited significant abnormalities in related biochemical markers, early symptoms of DMD, and multiple gray hair. His mother and sister were carriers with slightly abnormal biochemical markers. The mother had mild clinical symptoms, while the sister had no clinical symptoms. Other family members were genetically and physically normal. Sequencing and sequence alignment revealed that the inserted fragment was an Alu element from the AluYa5 subfamily. This insertion produced two stop codons and a polyadenylate (polyA) tail. To understand the impact of this insertion on the DMD gene and its association with clinical symptoms, exonic splicing enhancer (ESE) prediction indicated that the insertion did not affect the splicing of E59. Therefore, we speculated that the insertion sequence would be present in the mRNA sequence of the DMD gene. The two stop codons and polyA tail likely terminate translation, preventing the production of functional dystrophin protein, which may be the mechanism leading to DMD. In addition to typical DMD symptoms, the child also exhibited premature graying of hair. This study reports, for the first time, a case of DMD caused by the insertion of an Alu element into the coding region of the DMD gene. This finding provides clues for studying gene mutations induced by Alu sequence insertion and expands the understanding of DMD gene mutations.

Directed evolution of hyperactive integrases for site specific insertion of transgenes.

The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature.

A sequence of SVA retrotransposon insertions in ASIP shaped human pigmentation.

Retrotransposons comprise about 45% of the human genome1, but their contributions to human trait variation and evolution are only beginning to be explored2,3. Here, we find that a sequence of SVA retrotransposon insertions in an early intron of the ASIP (agouti signaling protein) gene has probably shaped human pigmentation several times. In the UK Biobank (n = 169,641), a recent 3.3-kb SVA insertion polymorphism associated strongly with lighter skin pigmentation (0.22 [0.21-0.23] s.d.; P = 2.8 × 10-351) and increased skin cancer risk (odds ratio = 1.23 [1.18-1.27]; P = 1.3 × 10-28), appearing to underlie one of the strongest common genetic influences on these phenotypes within European populations4-6. ASIP expression in skin displayed the same association pattern, with the SVA insertion allele exhibiting 2.2-fold (1.9-2.6) increased expression. This effect had an unusual apparent mechanism: an earlier, nonpolymorphic, human-specific SVA retrotransposon 3.9 kb upstream appeared to have caused ASIP hypofunction by nonproductive splicing, which the new (polymorphic) SVA insertion largely eliminated. Extended haplotype homozygosity indicated that the insertion allele has risen to allele frequencies up to 11% in European populations over the past several thousand years. These results indicate that a sequence of retrotransposon insertions contributed to a species-wide increase, then a local decrease, of human pigmentation.

Resistance mechanisms of EGFR tyrosine kinase inhibitors, in EGFR exon 20 insertion-mutant lung cancer.

BACKGROUND: Mobocertinib, an EGFR exon 20 insertion (Ex20ins)-specific tyrosine kinase inhibitor has been used for treatment of advanced/metastatic EGFR Ex20ins-mutant non-small cell lung cancer (NSCLC). However, resistance mechanisms to EGFR Ex20ins-specific inhibitors and the efficacy of subsequent amivantamab treatment is unknown. METHODS: To investigate resistance mechanisms, tissue and cfDNA samples were collected before treatment initiation and upon development of resistance from NSCLC patients with EGFR Ex20ins mutations received mobocertinib, poziotinib, and amivantamab treatments. Genetic alterations were analyzed using whole-genome and targeted sequencing, and in vitro resistant cell lines were generated for validation. RESULTS: EGFR amplification (n = 6, including 2 broad copy number gain) and EGFR secondary mutation (n = 3) were observed at the resistance of mobocertinib. One patient had both EGFR secondary mutation and high EGFR focal amplification. In vitro models harboring EGFR alterations were constructed to validate resistance mechanisms and identify overcoming strategies to resistance. Acquired EGFR-dependent alterations were found to mediate resistance to mobocertinib in patients and in vitro models. Furthermore, two of six patients who received sequential amivantamab followed by an EGFR tyrosine kinase inhibitor had MET amplification and showed partial response. CONCLUSIONS: Our study revealed EGFR-dependent and -independent mechanisms of mobocertinib resistance in patients with advanced EGFR Ex20ins-mutant NSCLC.

Identification of Two Potential Gene Insertion Sites for Gene Editing on the Chicken Z/W Chromosomes.

The identification of accurate gene insertion sites on chicken sex chromosomes is crucial for advancing sex control breeding materials. In this study, the intergenic region NC_006127.4 on the chicken Z chromosome and the non-repetitive sequence EE0.6 on the W chromosome were selected as potential gene insertion sites. Gene knockout vectors targeting these sites were constructed and transfected into DF-1 cells. T7E1 enzyme cleavage and luciferase reporter enzyme analyses revealed knockout efficiencies of 80.00% (16/20), 75.00% (15/20), and 75.00% (15/20) for the three sgRNAs targeting the EE0.6 site. For the three sgRNAs targeting the NC_006127.4 site, knockout efficiencies were 70.00% (14/20), 60.00% (12/20), and 45.00% (9/20). Gel electrophoresis and high-throughput sequencing were performed to detect potential off-target effects, showing no significant off-target effects for the knockout vectors at the two sites. EdU and CCK-8 proliferation assays revealed no significant difference in cell proliferation activity between the knockout and control groups. These results demonstrate that the EE0.6 and NC_006127.4 sites can serve as gene insertion sites on chicken sex chromosomes for gene editing without affecting normal cell proliferation.

A convenient five-segment cassette procedure for DNA insertions coding for novel peptides.

A DNA cassette assembly method is described which utilizes inexpensive oligomers no longer than 40 nt in length. The five-segment cassettes have 20 nt overlaps which give an effective length of 80 nt, making it possible to code for peptides up to 20 amino acids long. The cassettes have three phosphate free nicks, which can be successfully inserted into plasmid DNA and used to transform E. coli. The nicks are repaired in vivo by an unknown mechanism. Insertions are not successful for cassettes with greater than three nicks. A procedure is provided for rapid turnaround from DNA design to peptides, which are easily isolated as C-terminal fusions with GFP. The technique generally gives the expected sequence, with errors which occur about 1% of the time. Several representative DNA inserts are described which illustrate the method, as well as chemical details on the new peptides coded for. The peptides can be readily mutated to make it possible to understand how polar and aromatic residues affect GFP-fusion solubility, and how histidine residues can be strategically placed in a peptide for good IMAC retention. The method can be used to explore a large number of new designed peptides as fusion products quickly and economically.

Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species.

The canonical non-homologous end joining (c-NHEJ) repair pathway, generally viewed as stochastic, has recently been shown to produce predictable outcomes in CRISPR-Cas9 mutagenesis. This predictability, mainly in 1-bp insertions and small deletions, has led to the development of in-silico prediction programs for various animal species. However, the predictability of CRISPR-induced mutation profiles across species remained elusive. Comparing CRISPR-Cas9 repair outcomes between human and plant species reveals significant differences in 1-bp insertion profiles. The high predictability observed in human cells links to the template-dependent activity of human Polλ. Yet plant Polλ exhibits dual activities, generating 1-bp insertions through both templated and non-templated manners. Polλ knockout in plants leads to deletion-only mutations, while its overexpression enhances 1-bp insertion rates. Two conserved motifs are identified to modulate plant Polλ's dual activities. These findings unveil the mechanism behind species-specific CRISPR-Cas9-induced insertion profiles and offer strategies for predictable, precise genome editing through c-NHEJ.

A deoxyviolacein-based transposon insertion vector for pigmented tracer studies.

Pigments provide a simple means to rapidly visually ascertain the quantities or presence of specific microbes in a complex community. The selection of pigment-producing colonies that are simple to differentiate from common colony phenotypes provides a high degree of certainty for the identity of pigment-tagged strains. Successful employment of pigment production is dependent on various intrinsic factors related to proper levels of gene expression and pigment production that are not always easy to predict and vary within each microbe. We have constructed a simple transposon system that incorporates the genes for the production of deoxyviolacein, a pigment produced from intracellular reserves of the amino acid tryptophan, to randomly insert these genes throughout the genome. This tool allows the user to select from many thousands of potential sites throughout a bacterial genome for an ideal location to generate the desired amount of pigment. We have applied this system to a small selection of endophytes and other model bacteria to differentiate these strains from complex communities and confirm their presence after several weeks in natural environments. We provide two examples of applications using the pigments to trace strains following introduction into plant tissues or to produce a reporter strain for extracellular nitrogen compound sensing. We recognize that this tool could have far broader utility in other applications and microbes, and describe the methodology for use by the greater scientific community.

Exonic Short Interspersed Nuclear Element Insertion in FAM161A Is Associated with Autosomal Recessive Progressive Retinal Atrophy in the English Shepherd.

Progressive retinal atrophies (PRAs) are a genetically heterogeneous group of inherited eye diseases that affect over 100 breeds of dog. The initial clinical sign is visual impairment in scotopic conditions, as a consequence of rod photoreceptor cell degeneration. Photopic vision degeneration then follows, due to progression of the disease to the cone photoreceptors, and ultimately results in complete blindness. Two full-sibling English Shepherds were diagnosed with PRA at approximately 5 years old and tested clear of all published PRA genetic variants. This study sought to identify the novel PRA-associated variant segregating in the breed. We utilised a combined approach of whole genome sequencing of the probands and homozygosity mapping of four cases and 22 controls and identified a short interspersed nuclear element within an alternatively spliced exon in FAM161A. The XP_005626197.1 c.17929_ins210 variant was homozygous in six PRA cases and heterozygous or absent in control dogs, consistent with a recessive mode of inheritance. The insertion is predicted to extend exon 4 by 39 aberrant amino acids followed by an early termination stop codon. PRA is intractable to treatment, so the development of a genetic screening test, based on the associated variant, is significant, because it provides dog breeders/owners with a means of reducing the frequency of the disease variant within this breed as well as minimising the risk of breeding puppies that will develop this blinding disease.

A phase II study of osimertinib in patients with NSCLC harboring EGFR exon 20 insertion: A multicenter trial of the Korean Cancer Study Group (LU17-19).

BACKGROUND: Epidermal growth factor receptor (EGFR) exon 20 insertions account for up to 10% of all EGFR mutations. Clinical outcomes in patients receiving approved EGFR exon 20 insertion-specific inhibitors have been variable. Although osimertinib has demonstrated antitumor activity in clinical trials, its clinical efficacy and translational potential remain to be determined in non-small cell lung carcinoma (NSCLC) with EGFR exon 20 insertion. METHODS: In this multicenter phase II study, patients with advanced NSCLC harboring EGFR exon 20 insertions for whom the standard chemotherapy failed received 80 mg osimertinib once daily. The primary endpoint was the investigator-assessed objective response rate (ORR) as defined by Response Evaluation Criteria in Solid Tumors version 1.1. The secondary endpoints were progression-free survival (PFS), overall survival (OS), and safety profile. RESULTS: Among 15 patients enrolled at stage 1, the best response was most commonly disease stabilization (73.3 %), which did not meet the stage 1 threshold (objective response ≥ 2/15). As of data cutoff, two patients remained on the treatment. The median PFS and OS were 3.8 (95 % confidence interval [CI] = 1.7-5.5) months and 6.5 (95 % CI = 3.9-not reached) months, respectively. Adverse events (≥grade 3) were anemia, hypercalcemia, and pneumonia (13.3 % each), and asthenia, femur fracture, increased alkaline phosphate, hyperkalemia, bone pain, and azotemia (6.7 % each). Pre-existing EGFR C797S mutation detected in plasma limited the efficacy of osimertinib. CONCLUSION: Osimertinib at 80 mg once daily had limited efficacy and mostly showed disease stabilization with an acceptable safety profile in advanced NSCLC harboring EGFR exon 20 insertions. CLINICALTRIALS: govIdentifier: NCT03414814.

A common Alu insertion in the 3'UTR of TMEM106B is associated with risk of dementia.

INTRODUCTION: Sequence variants in TMEM106B have been associated with an increased risk of developing dementia. METHODS: As part of our efforts to generate a set of mouse lines in which we replaced the mouse Tmem106b gene with a human TMEM106B gene comprised of either a risk or protective haplotype, we conducted an in-depth sequence analysis of these alleles. We also analyzed transcribed TMEM106B sequences using RNA-seq data (AD Knowledge portal) and full genome sequences (1000 Genomes). RESULTS: We identified an AluYb8 insertion in the 3' untranslated region (3'UTR) of the TMEM106B risk haplotype. We found this AluYb8 insertion in every risk haplotype analyzed, but not in either protective haplotypes or in non-human primates. DISCUSSION: We conclude that this risk haplotype arose early in human development with a single Alu-insertion event within a unique haplotype context. This AluYb8 element may act as a functional variant in conferring an increased risk of developing dementia. HIGHLIGHTS: We conducted an in-depth sequence analysis of (1) a risk and (2) a protective haplotype of the human TMEM106B gene. We also analyzed transcribed TMEM106B sequences using RNA-seq data (AD Knowledge Portal) and full genome sequences (1000 Genomes). We identified an AluYb8 insertion in the 3' untranslated region (3'UTR) of the TMEM106B risk haplotype. We found this AluYb8 insertion in every risk haplotype analyzed, but not in either protective haplotypes or in non-human primates. This AluYb8 element may act as a functional variant in conferring an increased risk of developing dementia.

Loops are geometric catalysts for DNA integration.

The insertion of DNA elements within genomes underpins both genetic diversity and disease when unregulated. Most of DNA insertions are not random and the physical mechanisms underlying the integration site selection are poorly understood. Here, we perform Molecular Dynamics simulations to study the insertion of DNA elements, such as viral DNA or transposons, into naked DNA or chromatin substrates. More specifically, we explore the role of loops within the polymeric substrate and discover that they act as 'geometric catalysts' for DNA integration by reducing the energy barrier for substrate deformation. Additionally, we discover that the 1D pattern and 3D conformation of loops have a marked effect on the distribution of integration sites. Finally, we show that loops may compete with nucleosomes to attract DNA integrations. These results may be tested in vitro and they may help to understand patterns of DNA insertions with implications in genome evolution and engineering.

protoSpaceJAM: an open-source, customizable and web-accessible design platform for CRISPR/Cas insertional knock-in.

CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQR and enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce 'recoding' mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM's design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.

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.