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Heritable genome editing with CRISPR/Cas9 induces anosmia in a crop pest moth.

Koutroumpa FA, Monsempes C, François MC, de Cian A, Royer C, Concordet JP, Jacquin-Joly E - Sci Rep (2016)

Bottom Line: Lepidoptera suffer critical lack of genetic tools and heritable genome edition has been achieved only in a few model species.We find that 89.6% of the injected individuals carried Orco mutations, 70% of which transmitted them to the next generation.CRISPR/Cas9-mediated Orco knockout caused defects in plant odor and sex pheromone olfactory detection in homozygous individuals.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR iEES-Paris, route de Saint-Cyr, 78026 Versailles Cedex, France.

ABSTRACT
Lepidoptera suffer critical lack of genetic tools and heritable genome edition has been achieved only in a few model species. Here we demonstrate that the CRISPR/Cas9 system is highly efficient for genome editing in a non-model crop pest Lepidoptera, the noctuid moth Spodoptera littoralis. We knocked-out the olfactory receptor co-receptor Orco gene to investigate its function in Lepidoptera olfaction. We find that 89.6% of the injected individuals carried Orco mutations, 70% of which transmitted them to the next generation. CRISPR/Cas9-mediated Orco knockout caused defects in plant odor and sex pheromone olfactory detection in homozygous individuals. Our work genetically defines Orco as an essential OR partner for both host and mate detection in Lepidoptera, and demonstrates that CRISPR/Cas9 is a simple and highly efficient genome editing technique in noctuid pests opening new routes for gene function analysis and the development of novel pest control strategies.

No MeSH data available.


Related in: MedlinePlus

Comparison of the activity of the different guide RNAs (gRNAs).Three gRNAs (cr104, cr105, cr106) were co-injected with the Cas9 protein in different egg batches (9 batches illustrated for cr104 and 3 batches illustrated for each of the two other guides). Batches of emerging larvae were genotyped by the T7EI assay following genomic DNA extraction and PCR amplification of an Orco fragment (for fragment sequences and primers, see Fig. 1). DNA modification could be obtained only for cr104 and in all egg batches, as revealed by the gel pattern: the highest band corresponds to the wild type gDNA amplification and the two small bands (arrows) result from T7EI action. Water injected larvae (H2O) and no template (−) controls are shown for each primer combination. The ladder used is the Quick-load 2-Log DNA Ladder (0.1–10.0 kb, BioLabs).
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f2: Comparison of the activity of the different guide RNAs (gRNAs).Three gRNAs (cr104, cr105, cr106) were co-injected with the Cas9 protein in different egg batches (9 batches illustrated for cr104 and 3 batches illustrated for each of the two other guides). Batches of emerging larvae were genotyped by the T7EI assay following genomic DNA extraction and PCR amplification of an Orco fragment (for fragment sequences and primers, see Fig. 1). DNA modification could be obtained only for cr104 and in all egg batches, as revealed by the gel pattern: the highest band corresponds to the wild type gDNA amplification and the two small bands (arrows) result from T7EI action. Water injected larvae (H2O) and no template (−) controls are shown for each primer combination. The ladder used is the Quick-load 2-Log DNA Ladder (0.1–10.0 kb, BioLabs).

Mentions: Three gRNAs (cr104, cr105 and cr106) were designed (Fig. 1), injected in eggs and tested by genotyping pools of newly hatched larvae, to select the more efficient gRNA. PCR amplification of an Orco fragment encompassing the targeted sequence was followed by a T7 endonuclease I (T7EI) assay that recognized and cleaved heteroduplex DNA. By this assay, batches of larvae without any mutation revealed only one band corresponding to the wild type PCR amplified Orco fragment and batches of larvae containing mutations revealed two additional bands that resulted from T7EI action (Fig. 2). Out of the three gRNAs, only one was active at inducing mutations (cr104), and mutations were observed in all egg batches injected (Fig. 2). This gRNA was used further for individual studies by injection with Cas9 in 2569 eggs. In S. littoralis, eggs are laid in batches and percentages of injection survival (694 larvae hatched, 27%) ranged from 2.5% to 46% depending on the injected egg batch (Table 1). These percentages were similar to those observed for control eggs injected with water or just punctured (1.5% to 45%, 24% on average), suggesting that the protein/gRNA injected mix was not toxic. After individual genotyping, positive larvae were further reared until adults.


Heritable genome editing with CRISPR/Cas9 induces anosmia in a crop pest moth.

Koutroumpa FA, Monsempes C, François MC, de Cian A, Royer C, Concordet JP, Jacquin-Joly E - Sci Rep (2016)

Comparison of the activity of the different guide RNAs (gRNAs).Three gRNAs (cr104, cr105, cr106) were co-injected with the Cas9 protein in different egg batches (9 batches illustrated for cr104 and 3 batches illustrated for each of the two other guides). Batches of emerging larvae were genotyped by the T7EI assay following genomic DNA extraction and PCR amplification of an Orco fragment (for fragment sequences and primers, see Fig. 1). DNA modification could be obtained only for cr104 and in all egg batches, as revealed by the gel pattern: the highest band corresponds to the wild type gDNA amplification and the two small bands (arrows) result from T7EI action. Water injected larvae (H2O) and no template (−) controls are shown for each primer combination. The ladder used is the Quick-load 2-Log DNA Ladder (0.1–10.0 kb, BioLabs).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940732&req=5

f2: Comparison of the activity of the different guide RNAs (gRNAs).Three gRNAs (cr104, cr105, cr106) were co-injected with the Cas9 protein in different egg batches (9 batches illustrated for cr104 and 3 batches illustrated for each of the two other guides). Batches of emerging larvae were genotyped by the T7EI assay following genomic DNA extraction and PCR amplification of an Orco fragment (for fragment sequences and primers, see Fig. 1). DNA modification could be obtained only for cr104 and in all egg batches, as revealed by the gel pattern: the highest band corresponds to the wild type gDNA amplification and the two small bands (arrows) result from T7EI action. Water injected larvae (H2O) and no template (−) controls are shown for each primer combination. The ladder used is the Quick-load 2-Log DNA Ladder (0.1–10.0 kb, BioLabs).
Mentions: Three gRNAs (cr104, cr105 and cr106) were designed (Fig. 1), injected in eggs and tested by genotyping pools of newly hatched larvae, to select the more efficient gRNA. PCR amplification of an Orco fragment encompassing the targeted sequence was followed by a T7 endonuclease I (T7EI) assay that recognized and cleaved heteroduplex DNA. By this assay, batches of larvae without any mutation revealed only one band corresponding to the wild type PCR amplified Orco fragment and batches of larvae containing mutations revealed two additional bands that resulted from T7EI action (Fig. 2). Out of the three gRNAs, only one was active at inducing mutations (cr104), and mutations were observed in all egg batches injected (Fig. 2). This gRNA was used further for individual studies by injection with Cas9 in 2569 eggs. In S. littoralis, eggs are laid in batches and percentages of injection survival (694 larvae hatched, 27%) ranged from 2.5% to 46% depending on the injected egg batch (Table 1). These percentages were similar to those observed for control eggs injected with water or just punctured (1.5% to 45%, 24% on average), suggesting that the protein/gRNA injected mix was not toxic. After individual genotyping, positive larvae were further reared until adults.

Bottom Line: Lepidoptera suffer critical lack of genetic tools and heritable genome edition has been achieved only in a few model species.We find that 89.6% of the injected individuals carried Orco mutations, 70% of which transmitted them to the next generation.CRISPR/Cas9-mediated Orco knockout caused defects in plant odor and sex pheromone olfactory detection in homozygous individuals.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR iEES-Paris, route de Saint-Cyr, 78026 Versailles Cedex, France.

ABSTRACT
Lepidoptera suffer critical lack of genetic tools and heritable genome edition has been achieved only in a few model species. Here we demonstrate that the CRISPR/Cas9 system is highly efficient for genome editing in a non-model crop pest Lepidoptera, the noctuid moth Spodoptera littoralis. We knocked-out the olfactory receptor co-receptor Orco gene to investigate its function in Lepidoptera olfaction. We find that 89.6% of the injected individuals carried Orco mutations, 70% of which transmitted them to the next generation. CRISPR/Cas9-mediated Orco knockout caused defects in plant odor and sex pheromone olfactory detection in homozygous individuals. Our work genetically defines Orco as an essential OR partner for both host and mate detection in Lepidoptera, and demonstrates that CRISPR/Cas9 is a simple and highly efficient genome editing technique in noctuid pests opening new routes for gene function analysis and the development of novel pest control strategies.

No MeSH data available.


Related in: MedlinePlus