Transposon Insertion Sequencing Research Articles

In vitro exploration of the Xanthomonas hortorum pv. vitians genome using transposon insertion sequencing and comparative genomics to discriminate between core and contextual essential genes.

The essential genome of a bacterium encompasses core genes associated with basic cellular processes and conditionally essential genes dependent upon environmental conditions or the genetic context. Comprehensive knowledge of those gene sets allows for a better understanding of fundamental bacterial biology and offers new perspectives for antimicrobial drug research against detrimental bacteria such as pathogens. We investigated the essential genome of Xanthomonas hortorum pv. vitians, a gammaproteobacterial plant pathogen of lettuce (Lactuca sativa L.) which belongs to the plant-pathogen reservoir genus Xanthomonas and is affiliated to the family Xanthomonadaceae . No practical means of disease control or prevention against this pathogen is currently available, and its molecular biology is virtually unknown. To reach a comprehensive overview of the essential genome of X. hortorum pv. vitians LM16734, we developed a mixed approach combining high-quality full genome sequencing, saturated transposon insertion sequencing (Tn-Seq) in optimal growth conditions, and coupled computational analyses such as comparative genomics, synteny assessment and phylogenomics. Among the 370 essential loci identified by Tn-Seq, a majority was bound to critical cell processes conserved across bacteria. The remaining genes were either related to specific ecological features of Xanthomonas or Xanthomonadaceae species, or acquired through horizontal gene transfer of mobile genetic elements and associated with ancestral parasitic gene behaviour and bacterial defence systems. Our study sheds new light on our usual concepts about gene essentiality and is pioneering in the molecular and genomic study of X. hortorum pv. vitians.

A First Look at the Essential Genes of Pseudomonas protegens.

Transposon insertion sequencing is a useful tool to identify the genes that are essential for a bacterial species to grow and divide effectively. In this issue of Journal of Bacteriology, Fabian et al. present the first set of transposon insertion sequencing data highlighting the genes essential to the plant-commensal species Pseudomonas protegens strain Pf-5 and describe comparative analyses with other pseudomonads (B. K. Fabian, C. Foster, A. J. Asher, L. D. Elbourne, et al., J Bacteriol 203:e00432-20, 2021, https://doi.org/10.1128/JB.00432-20).

Elucidating Essential Genes in Plant-Associated Pseudomonas protegens Pf-5 Using Transposon Insertion Sequencing.

Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacterium that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used for this species. In this study, we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses, and transport were underrepresented. The majority of Pf-5 essential genes were part of the P. protegens core genome. Comparison of the essential gene set of Pf-5 with those of two plant-associated pseudomonads, P. simiae and P. syringae, and the well-studied opportunistic human pathogen P. aeruginosa PA14 showed that the four species share a large number of essential genes, but each species also had uniquely essential genes. Comparison of the Pf-5 in silico-predicted and in vitro-determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles may improve our understanding of the biological processes important for bacterial survival and growth.IMPORTANCE Essential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analyses of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of three other pseudomonads highlights how gene essentiality varies between closely related species.

Defined Mutant Library Sequencing (DML-Seq) for Identification of Conditional Essential Genes.

Transposon insertion sequencing (TIS) is an emerging technique which utilizes a massive transposon mutant library to screen specific phenotype and determine the conditional essential genetic requirements for bacterial fitness under distinct conditions combined with high-throughput parallel sequencing technology. Compared with a massive mutant library in traditional TIS, the defined mutant library sequencing (DML-Seq) has advantages as: 1) efficient mutagenesis; 2) low bottleneck effects; 3) avoid hotpots caused by screening; 4) can be directly used in the following experiments. Here, we described an optimized procedure of DML-Seq for fitness screen to supply classical TIS using the marine pathogenic bacterium Edwardsiella piscicida as an example.

Multiplexed Competition in a Synthetic Squid Light Organ Microbiome Using Barcode-Tagged Gene Deletions.

Beneficial symbioses between microbes and their eukaryotic hosts are ubiquitous and have widespread impacts on host health and development. The binary symbiosis between the bioluminescent bacterium Vibrio fischeri and its squid host Euprymna scolopes serves as a model system to study molecular mechanisms at the microbe-animal interface. To identify colonization factors in this system, our lab previously conducted a global transposon insertion sequencing (INSeq) screen and identified over 300 putative novel squid colonization factors in V. fischeri To pursue mechanistic studies on these candidate genes, we present an approach to quickly generate barcode-tagged gene deletions and perform high-throughput squid competition experiments with detection of the proportion of each strain in the mixture by barcode sequencing (BarSeq). Our deletion approach improves on previous techniques based on splicing by overlap extension PCR (SOE-PCR) and tfoX-based natural transformation by incorporating a randomized barcode that results in unique DNA sequences within each deletion scar. Amplicon sequencing of the pool of barcoded strains before and after colonization faithfully reports on known colonization factors and provides increased sensitivity over colony counting methods. BarSeq enables rapid and sensitive characterization of the molecular factors involved in establishing the Vibrio-squid symbiosis and provides a valuable tool to interrogate the molecular dialogue at microbe-animal host interfaces.IMPORTANCE Beneficial microbes play essential roles in the health and development of their hosts. However, the complexity of animal microbiomes and general genetic intractability of their symbionts have made it difficult to study the coevolved mechanisms for establishing and maintaining specificity at the microbe-animal host interface. Model symbioses are therefore invaluable for studying the mechanisms of beneficial microbe-host interactions. Here, we present a combined barcode-tagged deletion and BarSeq approach to interrogate the molecular dialogue that ensures specific and reproducible colonization of the Hawaiian bobtail squid by Vibrio fischeri The ability to precisely manipulate the bacterial genome, combined with multiplex colonization assays, will accelerate the use of this valuable model system for mechanistic studies of how environmental microbes-both beneficial and pathogenic-colonize specific animal hosts.

A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in Mycobacterium abscessus.

We present a technically simple, easy-to-perform method for generating the genomic libraries for Himar-1 transposon site sequencing (Tn-seq). In addition to being simpler than present methods in the technical aspect, it also allows more robust and straightforward identification of the insertion site, by generating a longer sequence surrounding the insertion TA in the genome. The method makes Tn-seq more user-friendly and accessible to laboratories with more-limited bioinformatic resources. Finally, we created a saturated transposon-mutant library in Mycobacterium abscessus and demonstrated the usefulness of the method in analysis of genes involved in colony morphology, as well as in analysis of the whole Tn-mutant library, with identification of over 8,000 unique mutants.IMPORTANCE Transposon insertion sequencing is a powerful tool, but many researchers are discouraged by the apparent technical complexity of preparing the genomic library for deep sequencing and by the complicated computational analysis needed for insertion site identification. Our proposed method makes the preparation of the library easy and straightforward, relying on well-known molecular biology techniques. In addition, the results obtained from the deep sequencing are easily analyzed in terms of transposon insertion site identification, placing library preparation and analysis within the reach of more researchers in the microbiology community, including those with less computational and bioinformatic resources and experience. This is demonstrated by analysis of the most saturated Tn-mutant library created to date in the emerging pathogen Mycobacterium abscessus.

Functional genomics reveals the toxin-antitoxin repertoire and AbiE activity in Serratia.

Bacteriophage defences are divided into innate and adaptive systems. Serratia sp. ATCC 39006 has three CRISPR-Cas adaptive immune systems, but its innate immune repertoire is unknown. Here, we re-sequenced and annotated the Serratia genome and predicted its toxin–antitoxin (TA) systems. TA systems can provide innate phage defence through abortive infection by causing infected cells to ‘shut down’, limiting phage propagation. To assess TA system function on a genome-wide scale, we utilized transposon insertion and RNA sequencing. Of the 32 TA systems predicted bioinformatically, 4 resembled pseudogenes and 11 were demonstrated to be functional based on transposon mutagenesis. Three functional systems belonged to the poorly characterized but widespread, AbiE, abortive infection/TA family. AbiE is a type IV TA system with a predicted nucleotidyltransferase toxin. To investigate the mode of action of this toxin, we measured the transcriptional response to AbiEii expression. We observed dysregulated levels of tRNAs and propose that the toxin targets tRNAs resulting in bacteriostasis. A recent report on a related toxin shows this occurs through addition of nucleotides to tRNA(s). This study has demonstrated the utility of functional genomics for probing TA function in a high-throughput manner, defined the TA repertoire in Serratia and shown the consequences of AbiE induction.

Vertical and Horizontal Transmission of ESBL Plasmid from Escherichia coli O104:H4.

Multidrug resistance (MDR) often results from the acquisition of mobile genetic elements (MGEs) that encode MDR gene(s), such as conjugative plasmids. The spread of MDR plasmids is founded on their ability of horizontal transference, as well as their faithful inheritance in progeny cells. Here, we investigated the genetic factors involved in the prevalence of the IncI conjugative plasmid pESBL, which was isolated from the Escherichia coli O104:H4 outbreak strain in Germany in 2011. Using transposon-insertion sequencing, we identified the pESBL partitioning locus (par). Genetic, biochemical and microscopic approaches allowed pESBL to be characterized as a new member of the Type Ib partitioning system. Inactivation of par caused mis-segregation of pESBL followed by post-segregational killing (PSK), resulting in a great fitness disadvantage but apparent plasmid stability in the population of viable cells. We constructed a variety of pESBL derivatives with different combinations of mutations in par, conjugational transfer (oriT) and pnd toxin-antitoxin (TA) genes. Only the triple mutant exhibited plasmid-free cells in viable cell populations. Time-lapse tracking of plasmid dynamics in microfluidics indicated that inactivation of pnd improved the survival of plasmid-free cells and allowed oriT-dependent re-acquisition of the plasmid. Altogether, the three factors—active partitioning, toxin-antitoxin and conjugational transfer—are all involved in the prevalence of pESBL in the E. coli population.

Functional characterization of asnC family transcriptional regulator in Pseudomonas aeruginosa PGPR2 during root colonization

Transcriptional regulators in bacteria are the crucial players in mediating communication between environmental cues and DNA transcription through a complex network process. Pseudomonas aeruginosa PGPR2 is an efficient root colonizer and a biocontrol strain. Previously, we identified that the transcriptional regulator, asnC, negatively regulates the corn root colonization of P. aeruginosa PGPR2. In a transposon insertion sequencing (INSeq) screen, the asnC insertion mutant was positively selected during root colonization, meaning the disruption of asnC improves the fitness of the P. aeruginosa PGPR2 strain for the root colonization. In this study, we constructed isogenic mutant of asnC family transcriptional regulator encoded by PGPR2_17510 by allele exchange mutagenesis. The ΔasnC mutant was able to efficiently colonize corn roots with a twofold increase in population when compared to the wild-type strain. Similarly, the mutant strain outcompeted the wild-type strain in a competition assay, where the mutant strain represented 90% of the total population recovered from the root. We compared the whole transcriptome of the wild-type and the ΔasnC mutant of P. aeruginosa PGPR2 when exposed to the corn root exudates. The RNA-Seq revealed that a total of 360 genes were differentially expressed in the ΔasnC strain of P. aeruginosa PGPR2. Inactivation of asnC transcriptional regulator resulted in the up-regulation of several genetic factors implicated in metabolism, uptake of nutrients, motility, stress response, and signal transduction, which could play crucial roles in root colonization. This notion was further validated by phenotypic characterization and quantification of transcription pattern of selected genes associated with metabolism, motility, and carbon catabolite repression between wild type and mutant strain, which was in agreement with transcriptome data. Similarly, ΔasnC strain formed increased biofilm on abiotic surface validating our RNA-seq analysis, where transcript levels of several genes associated with biofilm formation were up-regulated in the mutant strain. We report that the inactivation of an asnC family transcriptional regulator encoded by PGPR2_17510 enhances the root colonization and biofilm-forming ability of P. aeruginosa PGPR2. Together, our results provide evidence for the molecular adaptations that enable ΔasnC mutant strain to colonize on the corn roots and to form a biofilm.

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.

Lifestyle adaptations of Rhizobium from rhizosphere to symbiosis

By analyzing successive lifestyle stages of a model Rhizobium-legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N2-fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N2 Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N2 fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium-legume symbiosis.

Transposon Insertion Sequencing, a Global Measure of Gene Function.

The goal of genomics and systems biology is to understand how complex systems of factors assemble into pathways and structures that combine to form living organisms. Great advances in understanding biological processes result from determining the function of individual genes, a process that has classically relied on characterizing single mutations. Advances in DNA sequencing has made available the complete set of genetic instructions for an astonishing and growing number of species. To understand the function of this ever-increasing number of genes, a high-throughput method was developed that in a single experiment can measure the function of genes across the genome of an organism. This occurred approximately 10 years ago, when high-throughput DNA sequencing was combined with advances in transposon-mediated mutagenesis in a method termed transposon insertion sequencing (TIS). In the subsequent years, TIS succeeded in addressing fundamental questions regarding the genes of bacteria, many of which have been shown to play central roles in bacterial infections that result in major human diseases. The field of TIS has matured and resulted in studies of hundreds of species that include significant innovations with a number of transposons. Here, we summarize a number of TIS experiments to provide an understanding of the method and explanation of approaches that are instructive when designing a study. Importantly, we emphasize critical aspects of a TIS experiment and highlight the extension and applicability of TIS into nonbacterial species such as yeast.

Cell division is antagonized by the activity of peptidoglycan endopeptidases that promote cell elongation.

A peptidoglycan (PG) cell wall composed of glycans crosslinked by short peptides surrounds most bacteria and protects them against osmotic rupture. In Escherichia coli, cell elongation requires crosslink cleavage by PG endopeptidases to make space for the incorporation of new PG material throughout the cell cylinder. Cell division, on the contrary, requires the localized synthesis and remodeling of new PG at midcell by the divisome. Little is known about the factors that modulate transitions between these two modes of PG biogenesis. In a transposon-insertion sequencing screen to identify mutants synthetically lethal with a defect in the division protein FtsP, we discovered that mutants impaired for cell division are sensitive to elevated activity of the endopeptidases. Increased endopeptidase activity in these cells was shown to interfere with the assembly of mature divisomes, and conversely, inactivation of MepS was found to suppress the lethality of mutations in essential division genes. Overall, our results are consistent with a model in which the cell elongation and division systems are in competition with one another and that control of PG endopeptidase activity represents an important point of regulation influencing the transition from elongation to the division mode of PG biogenesis.

High-throughput discovery of phage receptors using transposon insertion sequencing of bacteria

As the most abundant microbes on Earth, novel bacteriophages (phages; bacteria-specific viruses) are readily isolated from environmental samples. However, it remains challenging to characterize phage-bacteria interactions, such as the host receptor(s) phages bind to initiate infection. Here, we tested whether transposon insertion sequencing (INSeq) could be used to identify bacterial genes involved in phage binding. As proof of concept, results showed that INSeq screens successfully identified genes encoding known receptors for previously characterized viruses of Escherichia coli (phages T6, T2, T4, and T7). INSeq screens were then used to identify genes involved during infection of six newly isolated coliphages. Results showed that candidate receptors could be successfully identified for the majority (five of six) of the phages; furthermore, genes encoding the phage receptor(s) were the top hit(s) in the analyses of the successful screens. INSeq screens provide a generally useful method for high-throughput discovery of phage receptors. We discuss limitations of our approach when examining uncharacterized phages, as well as usefulness of the method for exploring the evolution of broad versus narrow use of cellular receptors among phages in the biosphere.

Identification of genes that contribute to fitness of African and Global clades of Salmonella Enteritidis during infection of macrophages

Non-typhoidal Salmonella (NTS) usually cause gastroenteritis in humans, but in recent years NTS have begun to cause epidemics of bloodstream infections in Africa. Salmonella Enteritidis is the second most common serovar associated with this invasive form of NTS disease (iNTS) in Africa. To establish a systemic infection, Salmonella must survive and replicate within host cells, with macrophages being a primary target. Genomic characterisation of S. Enteritidis isolates from human bloodstream has identified two new clades that are unique to Africa and distinct from the Global Epidemic clade. The African S. Enteritidis clades exhibit genomic degradation, and possess a distinct prophage repertoire and are multi-drug resistant. However, little is known about the virulence factors that allow African S. Enteritidis to cause systemic infection in susceptible hosts. We screened libraries of random insertion mutants of African and Global S. Enteritidis by transposon insertion sequencing (TIS), and identified about 280 genes belonging to each clade that contribute to bacterial survival and replication in murine macrophages. The genes were associated with 5 pathogenicity-islands, or encoded the global regulators PhoPQ and OmpR-EnvZ. Experiments are ongoing to investigate the role in intra-macrophage replication of genes that are uniquely identified in African Salmonella. It is hoped that our findings will contribute to a greater understanding of African Salmonella infection biology, and that some of the virulence-associated genes could be potential targets for novel therapeutics.

MviN mediates the regulation of environmental osmotic pressure on esrB to control the virulence in the marine fish pathogen Edwardsiella piscicida

Edwardsiella piscicida is a notorious pathogen infecting diverse kinds of fish and causes substantial economic losses in the global aquaculture industries. The EsrA-EsrB two-component system plays a critical role in the regulation of virulence genes expression, including type III and type VI secretion systems (T3/T6SSs). In this study, the putative regulators of esrB were screened by the transposon insertion sequencing (TIS) technology. As a result, MviN, a lipid II flippase, was identified as a modulator to upregulate esrB and downstream T3/T6SS gene expression in the earlier growth phases while downregulate esrB at the later stages. Complement or overexpression of the mviN restored the esrB as well as T3/T6SS expression in the ΔmviN mutant strain. Moreover, MviN also mediated the regulation of environmental osmotic pressure on the expression of esrB. MviN was also found to significantly influence the in vivo colonization of E. piscicida in turbot. Collectively, this study enhanced our understanding of pathogenesis and virulence regulatory network of E. piscicida.

A decade of advances in transposon-insertion sequencing.

It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome-wide transposon mutagenesis with high-throughput sequencing to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome. Four TIS variations were published in 2009: transposon sequencing (Tn-Seq), transposon-directed insertion site sequencing (TraDIS), insertion sequencing (INSeq) and high-throughput insertion tracking by deep sequencing (HITS). TIS has since become an important tool for molecular microbiologists, being one of the few genome-wide techniques that directly links phenotype to genotype and ultimately can assign gene function. In this Review, we discuss the recent applications of TIS to answer overarching biological questions. We explore emerging and multidisciplinary methods that build on TIS, with an eye towards future applications.

Tn-Seq reveals hidden complexity in the utilization of host-derived glutathionein Francisellatularensis.

Host-derived glutathione (GSH) is an essential source of cysteine for the intracellular pathogen Francisella tularensis. In a comprehensive transposon insertion sequencing screen, we identified several F. tularensis genes that play central and previously unappreciated roles in the utilization of GSH during the growth of the bacterium in macrophages. We show that one of these, a gene we named dptA, encodes a proton-dependent oligopeptide transporter that enables growth of the organism on the dipeptide Cys-Gly, a key breakdown product of GSH generated by the enzyme γ-glutamyltranspeptidase (GGT). Although GGT was thought to be the principal enzyme involved in GSH breakdown in F. tularensis, our screen identified a second enzyme, referred to as ChaC, that is also involved in the utilization of exogenous GSH. However, unlike GGT and DptA, we show that the importance of ChaC in supporting intramacrophage growth extends beyond cysteine acquisition. Taken together, our findings provide a compendium of F. tularensis genes required for intracellular growth and identify new players in the metabolism of GSH that could be attractive targets for therapeutic intervention.

Genetic determinants of Pseudomonas aeruginosa fitness during biofilm growth

Pseudomonas aeruginosa is an environmental bacterium and an opportunistic human pathogen. It is also a well-established model organism to study bacterial adaptation to stressful conditions, such as those encountered during an infection process in the human host. Advancing knowledge on P. aeruginosa adaptation to biofilm growth conditions is bound to reveal novel strategies and targets for the treatment of chronic biofilm-associated infections. Here, we generated transposon insertion libraries in three P. aeruginosa strain backgrounds and determined the relative frequency of each insertion following biofilm growth using transposon sequencing. We demonstrate that in general the SOS response, several tRNA modifying enzymes as well as adaptation to microaerophilic growth conditions play a key role in bacterial survival under biofilm growth conditions. On the other hand, presence of genes involved in motility and PQS signaling were less important during biofilm growth. Several mutants exhibiting transposon insertions in genes detected in our screen were validated for their biofilm growth capabilities and biofilm specific transcriptional responses using independently generated transposon mutants. Our results provide new insights into P. aeruginosa adaptation to biofilm growth conditions. The detection of previously unknown determinants of biofilm survival supports the use of transposon insertion sequencing as a global genomic technology for understanding the establishment of difficult to treat biofilm-associated infections.

Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining.

One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.