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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

Sequences of the CRISPR/Cas9 induced mutations in the S. littoralis Orco gene obtained at G0.The PAM motif is highlighted as a grey box and the 20 bp cr104 guide RNA (gRNA) is shown with blue letters. The sequence modifications (Δ) are highlighted in red letters (insertions) and in red dashes (deletions). The sequences that were obtained from samples containing also sequence 1 are numbered with green numbers. The sequences with mutations that produced stop codons within the Orco coding sequence are designated with asterisks (*).
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f3: Sequences of the CRISPR/Cas9 induced mutations in the S. littoralis Orco gene obtained at G0.The PAM motif is highlighted as a grey box and the 20 bp cr104 guide RNA (gRNA) is shown with blue letters. The sequence modifications (Δ) are highlighted in red letters (insertions) and in red dashes (deletions). The sequences that were obtained from samples containing also sequence 1 are numbered with green numbers. The sequences with mutations that produced stop codons within the Orco coding sequence are designated with asterisks (*).

Mentions: All data described here are summarized in Table 1. Among the larvae that emerged from cr104/Cas9-injected eggs, 58 were individually genotyped, and 89.6% (52 larvae) of those presented mismatch at the Orco target region (T7EI gel assay). Thirty-three were sequenced, out of which 24 harboured multiple mutations. Due to sequence superposition in chromatograms, 17 of them could not be characterised. Seven (sequences 6, 7, 9, 10, 11, 13 and 14, Fig. 3) consisted of only two or three superposed sequences and could be easily characterised by visual curation of chromatograms. Further sequencing of G1 individuals that all carried a single mutated sequence confirmed the predicted sequences. All of the seven G0 superposed sequences were carrying the same background sequence, consisting of a 6 base pair (bp) deletion (sequence 1, Fig. 3). The IUPAC code was used when the superposing bases could not be clearly identified, typically when three sequences were superposed (Fig. 3). For nine larvae (out of 33, 27%), unique mutated Orco sequences were detected (sequences 1–5, 8, 12, 15 and 16 in Fig. 3). Sequence 1 corresponded to the 6 bp deletion. This frequent 6 bp deletion mutation (50%: 8 out of 16 characterized mutated larvae) does not induce stop codon and has possibly no consequence on protein function. Eleven mutated sequences resulted in frameshift in the open reading frame generating a premature stop codon (sequences 2–7, 9, 11, 14, 15 and 16 in Fig. 3), and are thus expected to generate non-functional Orco proteins.


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)

Sequences of the CRISPR/Cas9 induced mutations in the S. littoralis Orco gene obtained at G0.The PAM motif is highlighted as a grey box and the 20 bp cr104 guide RNA (gRNA) is shown with blue letters. The sequence modifications (Δ) are highlighted in red letters (insertions) and in red dashes (deletions). The sequences that were obtained from samples containing also sequence 1 are numbered with green numbers. The sequences with mutations that produced stop codons within the Orco coding sequence are designated with asterisks (*).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Sequences of the CRISPR/Cas9 induced mutations in the S. littoralis Orco gene obtained at G0.The PAM motif is highlighted as a grey box and the 20 bp cr104 guide RNA (gRNA) is shown with blue letters. The sequence modifications (Δ) are highlighted in red letters (insertions) and in red dashes (deletions). The sequences that were obtained from samples containing also sequence 1 are numbered with green numbers. The sequences with mutations that produced stop codons within the Orco coding sequence are designated with asterisks (*).
Mentions: All data described here are summarized in Table 1. Among the larvae that emerged from cr104/Cas9-injected eggs, 58 were individually genotyped, and 89.6% (52 larvae) of those presented mismatch at the Orco target region (T7EI gel assay). Thirty-three were sequenced, out of which 24 harboured multiple mutations. Due to sequence superposition in chromatograms, 17 of them could not be characterised. Seven (sequences 6, 7, 9, 10, 11, 13 and 14, Fig. 3) consisted of only two or three superposed sequences and could be easily characterised by visual curation of chromatograms. Further sequencing of G1 individuals that all carried a single mutated sequence confirmed the predicted sequences. All of the seven G0 superposed sequences were carrying the same background sequence, consisting of a 6 base pair (bp) deletion (sequence 1, Fig. 3). The IUPAC code was used when the superposing bases could not be clearly identified, typically when three sequences were superposed (Fig. 3). For nine larvae (out of 33, 27%), unique mutated Orco sequences were detected (sequences 1–5, 8, 12, 15 and 16 in Fig. 3). Sequence 1 corresponded to the 6 bp deletion. This frequent 6 bp deletion mutation (50%: 8 out of 16 characterized mutated larvae) does not induce stop codon and has possibly no consequence on protein function. Eleven mutated sequences resulted in frameshift in the open reading frame generating a premature stop codon (sequences 2–7, 9, 11, 14, 15 and 16 in Fig. 3), and are thus expected to generate non-functional Orco proteins.

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