Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis
Summary
This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.
Abstract
Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.
Introduction
Vector-borne diseases are a major public health threat in constant evolution. Hundreds of vector species spread across very distinct phylogenic families (e.g., mosquitoes, ticks, fleas) are responsible for the transmission of a huge number of microbial pathogens, resulting in more than 700,000 human deaths a year, according to the World Health Organization. Among vector insects, phlebotomine sand flies (Diptera, Psychodidae) constitute a vast group, with 80 proven vector species exhibiting distinct phenotypic traits and vectorial capacities found in different geographical regions. They are vectors for the protozoan parasites of the genus Leishmania, causing around 1.3 million new cases of Leishmaniases and between 20,000 and 30,000 deaths a year. Leishmaniases clinical outcomes are diverse, with symptoms ranging from self-limiting cutaneous lesions to visceral dissemination which is fatal in the absence of treatment.
Sand flies are strictly terrestrial insects. Their life cycle, relatively long compared to other Diptera, lasts up to three months, depending on different parameters such as temperature, humidity, and nutrition. It consists of one embryonic stage (6 to 11 days), four larval stages (lasting a total of 23 to 25 days) and one pupal stage (9 to 10 days) followed by metamorphosis and then adulthood. Sand flies require a humid and warm environment for rearing. Both males and females feed on sugars, obtained in the wild from flower nectars. Only females are blood-feeders, as they require proteins obtained from the blood meal for egg production1.
An important focus of research is to identify the nature of the vector/parasite interactions that lead to the development of transmissible infections. As with other vector insects, parameters intrinsic to sand flies have been shown to impact their vector competence, which is defined as their ability to carry and transmit pathogens to their hosts. For example, the expression of galectins by the Phlebotomus papatasi sand fly midgut cells, acting as receptors recognizing parasite surface components, can directly influence their vector competence for Leishmania major2,3. The insect immune response pathway, Immune Deficiency (IMD), is also crucial for the Phlebotomus papatasi sand fly vector competence for Leishmania major4. A critical role for vector insect immune response pathways in controlling their transmission of infectious pathogens has been similarly reported in Aedes aegypti mosquitos5,6,7, in the tsetse fly Glossina morsitans8, and in Anopheles gambiae mosquitoes9,10.
Studies of sand fly/Leishmania interactions have been limited by the lack of gene expression modification methods adapted for use in these insects. Only gene downregulation by small interfering RNA (siRNA) had been performed11,12,13,14 until recently. The technique, limited by the mortality associated with the microinjection of adult females, leads only to a partial loss of function, which cannot be transmitted from generation to generation.
CRISPR/Cas9 technology has revolutionized functional genomic research in non-model organisms such as sand flies. Modified from the adaptive immune system in prokaryotes for defense against bacteriophages15,16, the CRISPR Cas9 system has been rapidly adapted as a genome editing tool for superior eukaryotic organisms, including insects. The principle of CRISPR/Cas9 targeted genome-editing is based on the complementarity of a single guide RNA (sgRNA) to a specific genomic locus. The Cas9 nuclease binds to the sgRNA and creates a double-strand DNA (dsDNA) break in the genomic DNA where the sgRNA associates with its complementary sequence. The Cas9-sgRNA complex is guided to the target sequence by 17 to 20 complementary bases in the sgRNA to the chosen locus, the dsDNA break can then be repaired by two independent pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR)17. NHEJ repair involves a simple closure of the break but frequently leads to small insertion/deletion events. DNA repair through HDR uses a donor DNA molecule sharing homology with the target DNA as a template for repair. Insects possess both machineries.
CRISPR/Cas9 technology can generate mutations in a chosen locus, through the NHEJ repair pathway; or for more complex genome editing strategies, such as knock-ins or expression reporters, through the HDR pathway with an appropriate donor template. In sand flies, null mutant alleles of the immune response factor Relish were generated through NHEJ-mediated CRISPR in Phlebotomus papatasi4. Sand fly embryos were also injected in another study with a CRISPR/Cas9 mix targeting the gene encoding Yellow. Still, no adults carrying the mutation were produced18. We describe here a detailed method of sand fly targeted mutagenesis by NHEJ-mediated CRISPR/Cas9, with a particular focus on the embryo microinjection, a critical step of the protocol.
Protocol
Representative Results
Discussion
We present here a recently developed embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 in Phlebotomus papatasi sand flies. Embryo microinjection for insect genetic modification was developed in Drosophila in the mid-1980s21 and is now routinely used in a wide variety of insects. Other methods for delivery of genetic modification materials have been developed for use in insects, such as ReMOT20,21<s…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank Vanessa Meldener-Harrell for critical reading of the manuscript.
Materials
| Black Filter Paper 4.25CM PK100 | VWR | 28342-012 | Cut into rectangles that are approximately 46 X 22mm. These are placed between the slide and the coverslip and act as a moist base layer for the embryos during injection. |
| Coverslips | Fisher Scientific | 12-543A | |
| Dissecting Microscope | Any brand | For aligning embryos | |
| Glass slides | Fisher Scientific | 12-550-A3 | Base layer of the microinjection set up Figure 2A |
| Insect cage | custom made or several commercial options | polycarbonate cage for adults holding and mating Lawyer, Phillip, Mireille Killick-Kendrick, Tobin Rowland, Edgar Rowton, and Petr Volf. “Laboratory Colonization and Mass Rearing of Phlebotomine Sand Flies (Diptera, Psychodidae).” Parasite 24. Accessed August 6, 2020. https://doi.org/10.1051/parasite/2017041. | |
| Larval food | custom made | a mix of rabbit chow and rabbit feces Lawyer, Phillip, Mireille Killick-Kendrick, Tobin Rowland, Edgar Rowton, and Petr Volf. “Laboratory Colonization and Mass Rearing of Phlebotomine Sand Flies (Diptera, Psychodidae).” Parasite 24. https://doi.org/10.1051/parasite/2017041. | |
| Microcaps 100 ml | Drummond | 1-000-1000 | Used to back fill microinjection needles |
| Mouth aspirator | John W. Hock Company | Model 612 | mouth aspirator with HEPA filter |
| Olympus SZX12 | Olympus Life Sciences | Microinjection microscope | |
| Ovipots | Nalge company | ovipots are made from 125-ml or 500-ml straigh-sided plolypropylene jars modified by drilling 2.5cm holes in the bottom and filled with 1cm of plaster of Paris. Lawyer, Phillip, Mireille Killick-Kendrick, Tobin Rowland, Edgar Rowton, and Petr Volf. “Laboratory Colonization and Mass Rearing of Phlebotomine Sand Flies (Diptera, Psychodidae).” Parasite 24. Accessed August 6, 2020. https://doi.org/10.1051/parasite/2017041. | |
| Paint Brush 6-0 | Any Art Supply Company | n/a | Used for aligning embryos |
| Propionic acid | Sigma-Aldrich | 402907 | antifungal agent |
| Standard Glass Capillaries | World Precision Instruments | 1B100-3 | Used for making microinjection needles |
| Trio-MPC100 Controller and MP845 Manipulator | Sutter Instruments | Microinjection Controller and Micromanipulator |
Referências
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