Mutagenesis Screen Research Articles

Identification of a Coxiella burnetii outer membrane porin required for intracellular replication.

Coxiella burnetii is a gram-negative, obligate intracellular pathogen that causes human Q fever. Within host cells, C. burnetii proliferates in a spacious, acidic, lysosome-derived Coxiella-containing vacuole (CCV) by a process that requires the Dot/Icm type IVB secretion system to deliver effectors that manipulate host cell functions. A previous transposon mutagenesis screen identified the gene cbu0937 as being important for intracellular replication of C. burnetii. Here, the function of Cbu0937 was investigated. The cbu0937::Tn mutant had no detectable defect replicating in the axenic acidified citrate cysteine medium 2. Additionally, intracellular replication of the cbu0937::Tn mutant was not restored by co-infection of host cells with an isogenic wild-type strain of C. burnetii. Thus, the cbu0937::Tn mutant has a cell-intrinsic intracellular replication defect. Intracellular replication of the cbu0937::Tn mutant was restored by complementing the gene in trans with a plasmid encoding either untagged or an epitope-tagged version of Cbu0937. Analysis of the predicted structure of the Cbu0937 protein using AlphaFold revealed high similarity between Cbu0937 and several bacterial porins. Fractionation studies and surface labeling of C. burnetii producing a functional epitope-tagged protein indicated the localization of Cbu0937 to the bacterial outer membrane. From these data, we conclude that cbu0937 encodes a porin that plays an essential role in supporting C. burnetii intracellular replication, which likely involves the acquisition of an important metabolite in the CCV lumen.

Engineering a New Generation of Gene Editors: Integrating Synthetic Biology and AI Innovations.

CRISPR-Cas technology has revolutionized biology by enabling precise DNA and RNA edits with ease. However, significant challenges remain for translating this technology into clinical applications. Traditional protein engineering methods, such as rational design, mutagenesis screens, and directed evolution, have been used to address issues like low efficacy, specificity, and high immunogenicity. These methods are labor-intensive, time-consuming, and resource-intensive and often require detailed structural knowledge. Recently, computational strategies have emerged as powerful solutions to these limitations. Using artificial intelligence (AI) and machine learning (ML), the discovery and design of novel gene-editing enzymes can be streamlined. AI/ML models predict activity, specificity, and immunogenicity while also enhancing mutagenesis screens and directed evolution. These approaches not only accelerate rational design but also create new opportunities for developing safer and more efficient genome-editing tools, which could eventually be translated into the clinic.

Genetic characterization of the AHAS mutant line K4 with resistance to AHAS-inhibitor herbicides in rapeseed (Brassica napus L.)

It remains a great challenge to control weeds in rapeseed fields in China. Breeding herbicide-resistant rapeseed varieties and using corresponding herbicide formulations has become the most economical and effective way to control weeds in rapeseed field. Characterization of more herbicide-resistant genetic resources will provide opportunities for breeders to develop rapeseed herbicide-resistant varieties with good agronomic performance. Previously, we obtained the tribenuron methyl (TBM)-resistant mutant K4 from ZS9 (Brassica napus L.) through ethyl methyl sulfonate mutagenesis and TBM foliar-spray screening. In this study, the inheritance and molecular characterization of the mutant K4 are carried out. Genetic investigation indicated that the herbicide-resistance of the K4 was controlled by one dominant allele at a single nuclear gene locus. Molecular characterization showed that a single point substitution at position 535 from C to T in BnAHAS3 (BnAHAS3535T), which resulted in a mutation at point 179 in BnAHAS3. The K4 showed a certain degree of resistance to TBM, bensulfuron methyl, and monosulfon sodium, which were 50, 30, and 5 times that of ZS9, respectively. AHAS enzyme assay, structural analysis of AHAS proteins, affinity detection between TBM and BnAHAS3 by surface plasmon resonance analysis, and the transgenic experiment in Arabidopsis using BnAHAS3535T confirmed that BnAHAS3535T endow the K4 with herbicides resistance. In addition, an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles BnAHAS3535T. In conclusion, our research identified and characterized one novel mutative AHAS allele in B. napus and enriched genetic resource for developing herbicide-resistant rapeseed cultivars.

PPP2R1A mutations cause ATR inhibitor sensitivity in ovarian clear cell carcinoma

Identification of ARID1A/ATR synthetic lethality led to ATR inhibitor phase II trials in ovarian clear cell carcinoma (OCCC), a cancer of unmet need. Using multiple CRISPR-Cas9 mutagenesis and interference screens, we show that inactivation of protein phosphatase 2A (PP2A) subunits, including PPP2R1A, enhance ATRi sensitivity in ARID1A mutant OCCC. Analysis of a new OCCC cohort indicates that 52% possess oncogenic PPP2R1A p.R183 mutations and of these, one half possessed both ARID1A as well as PPP2R1A mutations. Using CRISPR-prime editing to generate new isogenic models of PPP2R1A mutant OCCC, we found that PPP2R1A p.R183W and p.R183P mutations cause ATRi-induced S phase stress, premature mitotic entry, genomic instability and ATRi sensitivity in OCCC tumour cells. p.R183 mutation also enhanced both in vitro and in vivo ATRi sensitivity in preclinical models of ARID1A mutant OCCC. These results argue for the assessment of PPP2R1A mutations as a biomarker of ATRi sensitivity.

Characterization of Euglena gracilis Mutants Generated by Long-Term Serial Treatment with a Low Concentration of Ethyl Methanesulfonate.

Euglena gracilis is a microalga that has great promise for the production of biofuels, functional foods, and bioactive compounds, and mutagenesis and effective screening methods are required to develop Euglena strains that have industrial use. Ethyl methanesulfonate (EMS) is a widely used mutagen, but is highly lethal to Euglena at typical concentrations. In the present study, low-concentration, long-time EMS exposure combined with serial treatment was introduced for generating Euglena mutants. We then used screening protocols to select cells with altered motility or pigmentation, and isolated two distinct strains of Euglena: Mutant 333 and Mutant 335. Mutant 333 showed increased motility but exhibited a decreased differentiation rate and reduced paramylon content (13.5%), making it unsuitable for industrial applications. However, Mutant 335, which had a deficiency of chlorophyll, had a high paramylon content (31.62%) and a mild and pleasant odor profile due to decreased concentrations of certain volatile compounds, with confirmation by GC-MS analysis. The Mutant 335 strain is suitable for the production of functional food products and renewable jet fuel.

The essential genome of Plasmodium knowlesi reveals determinants of antimalarial susceptibility.

Measures to combat the parasites that cause malaria have become compromised because of reliance on a small arsenal of drugs and emerging drug resistance. We conducted a transposon mutagenesis screen in the primate malaria parasite Plasmodium knowlesi, producing the most complete classification of gene essentiality in any Plasmodium spp. to date, with the resolution to define truncatable genes. We found conservation in the druggable genome between Plasmodium spp. and divergences in mitochondrial metabolism. Perturbation analyses with the frontline antimalarial artemisinin revealed modulators that both increase and decrease drug susceptibility. Our findings aid prioritization of drug and vaccine targets for the Plasmodium vivax clade and reveal mechanisms of resistance that can inform therapeutic development.

Axin-binding domain of glycogen synthase kinase 3β facilitates functional interactions with voltage-gated Na+channel Nav1.6.

Axin-binding domain of glycogen synthase kinase 3β facilitates functional interactions with voltage-gated Na+channel Nav1.6.

Transposon mediated functional genomic screening for BRAF inhibitor resistance reveals convergent Hippo and MAPK pathway activation events

Genotype-informed anticancer therapies such as BRAF inhibitors can show remarkable clinical efficacy in BRAF-mutant melanoma; however, drug resistance poses a major hurdle to successful cancer treatment. Many resistance events to targeted therapies have been identified, suggesting a complex path to improve therapeutics. Here, we showed the utility of a piggyBac transposon activation mutagenesis screen for the efficient identification of genes that are resistant to BRAF inhibition in melanoma. Although several forward genetic screens performed in the same context have identified a broad range of resistance genes that poorly overlap, an integrative analysis revealed a much smaller functional diversity of resistance mechanisms, including reactivation of the MAPK pathway, PI3K-AKT pathway, and Hippo pathway, suggesting that a relatively small number of therapeutic strategies might overcome resistance manifested by a large gene set. Moreover, we illustrated the pivotal role of the Hippo pathway effector TAZ (encoded by the WWTR1 gene) in mediating BRAF inhibition resistance through transcriptional regulation of receptor tyrosine kinases and through interactions with the E3 ubiquitin ligase NEDD4L.

Klebsiella pneumoniae employs a type VI secretion system to overcome microbiota-mediated colonization resistance

Microbial species must compete for space and nutrients to persist in the gastrointestinal (GI) tract, and our understanding of the complex pathobiont-microbiota interactions is far from complete. Klebsiella pneumoniae, a problematic, often drug-resistant nosocomial pathogen, can colonize the GI tract asymptomatically, serving as an infection reservoir. To provide insight on how K. pneumoniae interacts with the resident gut microbiome, we conduct a transposon mutagenesis screen using a murine model of GI colonization with an intact microbiota. Among the genes identified were those encoding a type VI secretion system (T6SS), which mediates contact-dependent killing of gram-negative bacteria. From several approaches, we demonstrate that the T6SS is critical for K. pneumoniae gut colonization. Metagenomics and in vitro killing assays reveal that K. pneumoniae reduces Betaproteobacteria species in a T6SS-dependent manner, thus identifying specific species targeted by K. pneumoniae. We further show that T6SS gene expression is controlled by several transcriptional regulators and that expression only occurs in vitro under conditions that mimic the gut environment. By enabling K. pneumoniae to thrive in the gut, the T6SS indirectly contributes to the pathogenic potential of this organism. These observations advance our molecular understanding of how K. pneumoniae successfully colonizes the GI tract.

PRDM1 is a key regulator of the natural killer T-cell central memory program and effector function.

Natural killer T cells (NKTs) are a promising platform for cancer immunotherapy, but few genes involved in regulation of NKT therapeutic activity have been identified. To find regulators of NKT functional fitness, we developed a CRISPR/Cas9-based mutagenesis screen that employs a guide RNA (gRNA) library targeting 1,118 immune-related genes. Unmodified NKTs and NKTs expressing a GD2-specific chimeric antigen receptor (GD2.CAR) were transduced with the gRNA library and exposed to CD1d+ leukemia or CD1d-GD2+ neuroblastoma cells, respectively, over six challenge cycles in vitro. Quantification of gRNA abundance revealed enrichment of PRDM1-specific gRNAs in both NKTs and GD2.CAR NKTs, a result that was validated through targeted PRDM1 knockout. Transcriptional, phenotypic, and functional analyses demonstrated that CAR NKTs with PRDM1 knockout underwent central memory-like differentiation and resisted exhaustion. However, these cells downregulated the cytotoxic mediator granzyme B and showed reduced in vitro cytotoxicity and only moderate in vivo antitumor activity in a xenogeneic neuroblastoma model. In contrast, shRNA-mediated PRDM1 knockdown preserved effector function while promoting central memory differentiation, resulting in GD2.CAR NKTs with potent in vivo antitumor activity. Thus, we have identified PRDM1 as a regulator of NKT memory differentiation and effector function that can be exploited to improve the efficacy of NKT-based cancer immunotherapies.

An exacerbated phosphate starvation response triggers Mycobacterium tuberculosis glycerol utilization at acidic pH.

The mechanisms controlling Mycobacterium tuberculosis (Mtb) replication and survival inside its human host remain ill-defined. Phagosome acidification and nutrient deprivation are common mechanisms used by macrophages to restrict the replication of intracellular bacteria. Mtb stops replicating at mildly acidic pH (<pH5.8), an adaptive process called "acid growth arrest," which is thought to play an important role in Mtb virulence. Using a genome-wide mutagenesis screen, we identified 95 genes whose disruption either decreases or increases Mtb fitness during acid growth arrest. We show that the virulence-associated inorganic phosphate (Pi) uptake system (Pst-1) regulates the ability of Mtb to replicate in acidic conditions. Deletion of pstA1, a gene coding for a subunit of the Pst-1 system, results in the overexpression of the Pi starvation regulator regX3, which is sufficient to restore Mtb growth in acid conditions. Our data further support the role of limited glycerol uptake and ROS-mediated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition in causing Mtb acid growth arrest. This study reveals an unexpected role of the Pi starvation response in regulating acid growth arrest and highlights the intricacy of the mechanisms regulating redox homeostasis, acid stress response, and nutrient utilization in Mtb.IMPORTANCEDespite the availability of antibiotic treatment, M. tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains a major infectious disease killer worldwide. A better understanding of the environments that Mtb faces during infection and the mechanisms Mtb employs to respond and adapt may help identify currently unexplored pathways and targets for the development of novel anti-TB drugs. Here, we demonstrate that Mtb growth in acid can be restored by the over-expression of the Pi starvation response regulator regX3. This work paves the way toward a better understanding of the mechanisms controlling Mtb growth at acidic pH and highlights the role of inorganic phosphate in this process.

Computational design and improvement of a broad influenza virus HA stem targeting antibody.

Computational design and improvement of a broad influenza virus HA stem targeting antibody.

Enhancing the erythritol production of Yarrowia lipolytica by high-throughput screening based on highly sensitive artificial sensor and anchor protein cwp2.

In this study, we constructed an artificial sensor system for effective erythritol detection, established a single-cell droplet-based high-throughput screening system based on fluorescence-activated cell sorting, and induced and screened mutant strains of Yarrowia lipolytica with improved erythritol production through mutagenesis and high-throughput screening.

The histone demethylase Kdm5 controls Hid-induced cell death in Drosophila

We conducted an EMS mutagenesis screen on chromosome arm 2L to identify recessive suppressors of GMR-hid-induced apoptosis in the Drosophila eye. Through this screen, we recovered three alleles of the lysine demethylase gene Kdm5. Kdm5, a member of the JmjC-domain-containing protein family, possesses histone demethylase activity towards H3K4me3. Our data suggest that Kdm5 specifically regulates Hid-induced cell death during development, as we did not observe control of Reaper- or Grim-induced cell death by Kdm5. Interestingly, GMR-hid-induced apoptosis is suppressed independently of Kdm5’s demethylase activity. Our findings indicate that Rbf and dMyc are necessary for Kdm5 mosaics to suppress GMR-hid-induced cell death. Moreover, Kdm5 mosaics failed to suppress apoptosis induced by a mutant form of Hid that is resistant to inhibition by Erk-type MAPK activity. Additionally, Kdm5 dominantly enhances the wing phenotype of an activated MAPK mutant. These results collectively suggest that Kdm5 controls Hid-induced apoptosis by regulating the Rbf, dMyc, and MAPK pathways.

Comparative analysis and directed protein evolution yield an improved degron technology with minimal basal degradation, rapid inducible depletion, and faster recovery of target proteins.

Biological mechanisms are inherently dynamic, requiring precise and rapid gene manipulation for effective characterization. Traditional genetic perturbation tools such as siRNA and CRISPR knockout operate on timescales that render them unsuitable for exploring dynamic processes or studying essential genes, where chronic depletion can lead to cell death. Here, we compared four major inducible degron systems-dTAG, HaloPROTAC, and two auxin-inducible degron (AID) tools-in human pluripotent stem cells. We evaluated basal degradation levels, inducible degradation kinetics, and recovery dynamics for endogenously tagged genes. While the AID 2.0 system is the most efficient for rapid protein degradation, it exhibited higher basal degradation and slower recovery after ligand washout. To address these challenges, we applied directed protein evolution, incorporating base-editing-mediated mutagenesis and iterative functional selection and screening. We discovered novel OsTIR1 variants, including S210A, with significantly enhanced overall degron efficiency. The resulting system, designated as AID 3.0, demonstrates minimal basal degradation and rapid and effective target protein depletion and substantially rescues the cellular and molecular phenotypes due to basal degradation or slow target protein recovery in previous systems. We conclude that AID 3.0 represents a superior degron technology, offering a valuable tool for studying gene functions in dynamic biological contexts and exploring therapeutic applications. Additionally, the research strategy used here could be broadly applicable for improving other degron and biological tools.

Enhanced lutein and protein production with improved organoleptic properties in a novel yellow strain of Auxenochlorella pyrenoidosa mutant through atmospheric and room temperature plasma mutagenesis and norflurazon-based screening

Enhanced lutein and protein production with improved organoleptic properties in a novel yellow strain of Auxenochlorella pyrenoidosa mutant through atmospheric and room temperature plasma mutagenesis and norflurazon-based screening

Structural insights into the mechanotransducing mechanism of FtsEX in cell division.

The filamentous temperature-sensitive (Fts) protein FtsEX plays a pivotal role in Escherichia coli (E. coli) cell division by facilitating the activation of peptidoglycan hydrolysis through the adaptor EnvC. FtsEX belongs to the type VII ATP-binding cassette (ABC) transporter superfamily, which harnesses ATP energy to induce mechanical force, triggering a cascade of conformational changes that activate the pathway. However, the precise mechanism by which FtsEX initiates mechanotransmission remains elusive. Due to the inherent instability of this type of ABC transporter protein in vitro, the conformation of FtsEX has solely been determined in the stabilized ATP-bound state. To elucidate the dynamics of FtsEX, we characterized FtsEX and EnvC of various functional structures through cryo-electron microscopy (cryo-EM) and homology modeling. We validated the structures by molecular dynamics simulations. By site-directed mutagenesis and phenotype screening, we also identified the functional residues involved in allosteric communication between FtsE and FtsX as well as FtsX and EnvC. Additionally, we discovered a potential role of phospholipids in stabilizing the complex conformation during mechanotransmission. This comprehensive exploration significantly enhances our understanding of the intricate mechanisms governing bacterial cell division and unveils potential molecular targets for developing innovative antimicrobial drugs to combat antibiotic resistance.

Base editing screens define the genetic landscape of cancer drug resistance mechanisms

Drug resistance is a principal limitation to the long-term efficacy of cancer therapies. Cancer genome sequencing can retrospectively delineate the genetic basis of drug resistance, but this requires large numbers of post-treatment samples to nominate causal variants. Here we prospectively identify genetic mechanisms of resistance to ten oncology drugs from CRISPR base editing mutagenesis screens in four cancer cell lines using a guide RNA library predicted to install 32,476 variants in 11 cancer genes. We identify four functional classes of protein variants modulating drug sensitivity and use single-cell transcriptomics to reveal how these variants operate through distinct mechanisms, including eliciting a drug-addicted cell state. We identify variants that can be targeted with alternative inhibitors to overcome resistance and functionally validate an epidermal growth factor receptor (EGFR) variant that sensitizes lung cancer cells to EGFR inhibitors. Our variant-to-function map has implications for patient stratification, therapy combinations and drug scheduling in cancer treatment.

Emc1 is essential for vision and zebrafish photoreceptor outer segment morphogenesis.

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1-/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1-/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated.

Breeding of L-tryptophan high-yield strain by ARTP mutagenesis and high-throughput screening

L-tryptophan is an indispensable essential amino acid with a wide range of applications, which leads to a high demand. Accordingly, the production of L-tryptophan becomes a much-anticipated direction in research and industrial development. While irrational mutagenesis is an effective means to breed industrial strains, how to screen the strains with desirable phenotypes is still a major challenge. In order to improve the efficiency and accuracy of screening L-tryptophan high-yield strains, we used atmospheric and room temperature plasma mutagenesis to construct a random mutant library and then combined it with high-throughput screening in deep-well plates. Using a pseudo-fluorescent protein sensor capable of responding specifically to L-tryptophan, we successfully screened out a strain producing L-tryptophan at a high yield from a random mutagenesis library. The fermentation with the strain in shake flasks produced L-tryptophan at a yield of 1.99 g/L, which was 41.77% higher than that of the starting strain. Finally, the mechanism of high yield of the strain was deciphered by comparative genomics and transcriptomics. The above strategies provide a solid research foundation for further selection and development of high quality L-tryptophan producing strains.