Biology subjects

Pines, G.

Publications and source records attributed to Pines, G..

8 recordsLinked to original sources

bioRxiv2025

An OpIE2-DsRed marker disrupts female blood-feeding and shortens lifespan in the malaria vector Anopheles gambiae.

Anopheles gambiae is one of the principal vectors of human malaria. Over the past two decades, transgenic mosquito strains have been essential tools for studying mosquito biology and developing genetic control strategies such as gene drives. Mosquito transformants are typically identified using fluorescent markers, which are assumed to be phenotypically neutral. While generating CRISPR-based gene drive strains carrying an OpIE2-DsRed marker we unexpectedly found that transgenic females were unable to blood-feed and were consequently sterile, whereas males initially appeared normal and fertile. Given the potential utility of dominant, female-specific sterility for mosquito control, we established additional strains controlling for transgene content and integration site, confirming that the OpIE2-DsRed cassette caused the defect. Behavioral assays showed that females exhibited normal attraction to a membrane feeder but failed to initiate blood-feeding, performing repeated cycles of probing and proboscis grooming in rapid succession before ultimately leaving the feeder unfed. Microscopy showed that both sexes possessed a distally curved proboscis, providing a morphological explanation for the blood-feeding defect of females and the reduced male lifespan. A second promoter variant (OpIE2b), differing in flanking sequences at the IE-2 junction, drove strong marker expression without impairing blood-feeding or longevity. These findings demonstrate that minor differences in promoter architecture can produce major, unexpected phenotypic effects. OpIE2b provides a robust, phenotypically neutral marker for An. gambiae research, while OpIE2a highlights the need for rigorous validation of transgenic components intended for research and applied releases.

genetics↗

bioRxiv2025

CRISPR/Cas9-mediated mutagenesis of the white-eye gene in the tephritid pest Bactrocera zonata

Bactrocera zonata is a highly invasive agricultural pest that causes extensive damage to fruit crops. The Sterile Insect Technique (SIT), a species-specific and environmentally friendly pest control method, depends on the availability of Genetic Sexing Strains (GSSs) to enable efficient mass production of males for sterile release. However, no GSS currently exists for B. zonata limiting SIT applications targeting this important invasive pest. Here, we report two key advancements toward GSS development in this species. First, we present a high-quality, chromosome-level genome assembly from male B. zonata, identifying two scaffolds derived from the Y chromosome, which represent potential targets for future male-specific genetic engineering. Second, we demonstrate the feasibility of CRISPR/Cas9 genome editing in B. zonata by generating stable, homozygous white-eye mutants through targeted disruption of the conserved white-eye gene. This visible, recessive phenotype serves as a proof-of-concept for developing selectable markers in this species. Together, these results provide foundational genomic and genetic tools to support the development of GSSs in B. zonata, advancing the potential for sustainable, genetics-based pest control strategies.

bioengineering↗

bioRxiv2024

Rapid, direct, and sequence-specific identification of RNA viruses in various crop plants using CRISPR/Cas13a

Plant viruses are destructive pathogens causing significant damage to various crop species. Rapid, sensitive, and specific detection is crucial for the effective containment of emerging and resistance-breaking viruses. CRISPR/Cas has been established as a useful tool for plant virus identification. However, its application for on-site, direct detection of viruses from plant tissues is still limited. In this study, we present a rapid method for detecting viruses directly from RNA of different crop species using CRISPR/Cas13a. We successfully applied this method to identify tomato brown rugose fruit virus (ToBRFV) in infected tomato plants and differentiate it from closely related tobamoviruses. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Moreover, CRISPR/Cas13a was used to directly identify cucumber green mottle mosaic virus (CGMMV) in cucumber plants and turnip mosaic virus (TuMV) in Brassica napus plants. Finally, we developed a user-friendly, extraction-free, 15-minute protocol for on-site ToBRFV identification using a portable fluorescent viewer and a mobile phone camera. This protocol was successfully applied for ToBRFV detection in a commercial greenhouse. These results demonstrate that CRISPR/Cas13a is a robust technology for direct, rapid, sensitive, and specific identification of multiple viruses in different crop plants that can be easily implemented for on-site detection.

plant biology↗

bioRxiv2023

CRISPR-Based Genome Editing in Harmonia Axyridis

Harmonia axyridis (Pallas), commonly known as the Asian lady beetle, is a native insect species of Asia that has been intentionally introduced to various regions for biocontrol purposes. However, its widespread presence beyond its original release sites suggests a high degree of invasiveness. Previous studies have employed double-stranded RNA techniques to investigate the functional genomics of this species. In this study, we utilized the CRISPR-Cas9 approach to achieve precise genome editing in H. axyridis. Specifically, we targeted two distinct genes known to exhibit visible phenotypic effects. While the knockout of the laccase2 gene resulted in an early-detectable phenotype but also in lethality, we successfully established a viable and genetically stable mutant colony by disrupting the scarlet gene. Our findings contribute to the expanding knowledge of genetic manipulation in H. axyridis and provide insights into its potential for future research and practical applications for biocontrol and invasive species management.

genomics↗

bioRxiv2022

Rapid and sensitive on-site genetic diagnostics of pest fruit flies using CRISPR-Cas12a

Bactrocera zonata, a major fruit pest species, is gradually spreading west from its native habitat in East Asia. In recent years it has become a major threat to the Mediterranean area, with the potential of invading Europe, the Americas, and Australia. To prevent its spreading, monitoring efforts in plantation sites and border controls are carried out. Despite these efforts, and due to morphological similarities between B. zonata and other pests in relevant developmental stages, the monitoring process is challenging, time-consuming, and requires external assistance from professional labs. CRISPR-Cas12a genetic diagnostics has been rapidly developing in recent years and provides an efficient tool for the genetic identification of pathogens, viruses, and other genetic targets. Here we design a CRISPR-Cas12a detection assay that differentially detects two major pest species, B. zonata and Ceratitis capitata. Our easy-to-use and affordable assay employs a simple DNA extraction technique together with isothermal amplification, and Cas12a-based detection. We demonstrate the specificity and high sensitivity of this method, and its relevance for on-site applications. This method is highly modular, and the presented target design method can be applied to a wide array of pests. Key MassageO_LIDistinguishing different pest fruit flies on-site is crucial for prevention of global spreading but can be difficult C_LIO_LIWe present a genetic identification assay for rapid, on-site detection of pest using CRISPR-Cas12a C_LIO_LIThe method is affordable, quick and easy-to-use, and can be applied in border controls or on-site C_LIO_LIThe design process can be easily tailored for any pest, and can greatly benefit developing countries C_LI

synthetic biology↗

bioRxiv2021

Differential detection of Tomato mosaic virus (ToMV) and Tomato brown rugose fruit virus (ToBRFV) using CRISPR-Cas12

CRISPR/Cas12-based detection is a novel approach for efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the Tomato brown rugose fruit virus (ToBRFV), a new and emerging Tobamovirus causing substantial damage to the global tomato industry. Specific guide RNAs (gRNAs) were designed to detect either ToBRFV or the closely related Tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15-30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method enabled the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of plant viruses. This could provide a platform for the development of efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.

molecular biology↗

bioRxiv2018

Isolation of Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to Fosmidomycin

Sequence to activity mapping technologies are rapidly developing, enabling the isolation of mutations that confer novel phenotypes. Here we used the CRISPR EnAbled Trackable genome Engineering (CREATE) technology to investigate the inhibition of the essential IspC gene in Escherichia coli. IspC gene product, Deoxyxylulose Phosphate Reductoisomerase (DXR), converts 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate in the DXP pathway. Since this pathway is shared with many pathogenic bacteria and protozoa and is missing in humans, it is an appealing target for inhibition. We created a full saturation library of 33 sites proximal to ligand binding and other sites and challenged it with the DXR-specific inhibitor, fosmidomycin. We identified several mutations that confer fosmidomycin resistance. All sites are highly conserved and also exist in pathogens including the malaria-inducing Plasmodium falciparum. These findings may have general implications on the isolation of resistance-conferring mutations and specifically, may affect the design of future generations of fosmidomycin-based drugs.\n\nSignificanceThe emergence of acquired drug resistance is a natural process that is likely to occur under most circumstances. Recently-developed technologies allow to map relative fitness contribution of multiple mutations in parallel. Such approaches may be used to predict which mutations are most likely to confer resistance, instead of waiting for them to evolve spontaneously. In this study, a rationally-designed IspC mutant library was generated genomically in E. coli. Mutants resistant to fosmidomycin, an antimalarial drug were identified, and most were in the highly conserved proline at position 274. These results may have implications on next-generation fosmidomycin drug design, and more broadly, this approach may be used for predicting mutational acquired resistance.

microbiology↗

bioRxiv2017

Refactoring the Genetic Code for Increased Evolvability

The standard genetic code is robust to mutations and base-pairing errors during transcription and translation. Point mutations are most likely to be synonymous or preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best performing mutant requires a replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyper-evolvable organism serving as an ideal platform for directed evolution experiments. We then conclude by evaluating potential applications for recoded organisms within the synthetic biology field.\n\nSignificance StatementThe conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene using common error-prone methods. Here we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transition to a larger pool of amino acids and with a greater degree of chemical differences, using a single nucleotide replacement within the codon, thus increasing evolvability both at the single gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation and fitness maximization.

bioengineering↗