Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

Martín-Martín, Inés; Aryan, Azadeh; Meneses, Claudio; Adelman, Zach N.; Calvo, Eric

doi:10.1371/journal.pntd.0006769

Cited by 16 publications

(10 citation statements)

References 57 publications

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“…To our knowledge, no successful CRISPR/Cas9 mutagenesis in sand flies has been reported. Sand fly embryos were injected with a CRISPR/Cas9 mix targeting the gene encoding Yellow, but no adults carrying the mutation were produced (28). In this study, we adapted the CRISPR/Cas9 technique to Phlebotomus papatasi sand flies, a natural vector for L. major.…”

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confidence: 99%

CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major

Louradour

Ghosh

Inbar

et al. 2019

mBio

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Sand flies are the natural vectors for the Leishmania species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/Leishmania interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the Phlebotomus papatasi sand fly, a natural vector for Leishmania major, targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in P. papatasi and were able to generate transmissible null mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with L. major. Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for Leishmania parasites. IMPORTANCE Sand flies are the natural vectors of Leishmania parasites. Studies of sand fly/Leishmania interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible null mutant alleles of relish, a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with L. major, showing that the sand fly immune response impacts its vector competence for this pathogen.

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confidence: 99%

CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major

Louradour

Ghosh

Inbar

et al. 2019

mBio

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“…longipalpis genome in VectorBase ( , Table II), as well as their importance in the literature. 42 , 43 In addition, identification with a score greater than 50 reflects the quality of the peptide sequences obtained and the consequent identification of the protein, in the studied condition. These three proteins had their corresponding nucleic acid sequences analysed for the design of specific RT-PCR primers ( Table ).…”

Section: Resultsmentioning

confidence: 99%

Insights into the proteomic profile and gene expression of Lutzomyia longipalpis-derived Lulo cell line

Côrtes

Pita-Pereira

Farani

et al. 2020

Mem. Inst. Oswaldo Cruz

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BACKGROUND Lutzomyia longipalpis -derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania . OBJECTIVES Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis . METHODS Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

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“…To introduce a Wolbachia strain or perform genetic modifications, a protocol for the microinjection of biting midge eggs must be established. While there are well-established protocols for microinjecting mosquito eggs [ 74 ] and Drosophila and sandfly eggs [ 75 ], Culicoides eggs are more elongated and have less volume, and are thus anticipated to be more difficult to manipulate (Fig. 3 ).…”

Section: Research Gaps Concerning the Use Of Novel Control Approachesmentioning

confidence: 99%

Next-generation tools to control biting midge populations and reduce pathogen transmission

et al. 2021

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Biting midges of the genus Culicoides transmit disease-causing agents resulting in a significant economic impact on livestock industries in many parts of the world. Localized control efforts, such as removal of larval habitat or pesticide application, can be logistically difficult, expensive and ineffective if not instituted and maintained properly. With these limitations, a population-level approach to the management of Culicoides midges should be investigated as a means to replace or supplement existing control strategies. Next-generation control methods such as Wolbachia- and genetic-based population suppression and replacement are being investigated in several vector species. Here we assess the feasibility and applicability of these approaches for use against biting midges. We also discuss the technical and logistical hurdles needing to be addressed for each method to be successful, as well as emphasize the importance of addressing community engagement and involving stakeholders in the investigation and development of these approaches.

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Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

Cited by 16 publications

References 57 publications

CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major

CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major

Insights into the proteomic profile and gene expression of Lutzomyia longipalpis-derived Lulo cell line

Next-generation tools to control biting midge populations and reduce pathogen transmission

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