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Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies.

Itokawa K, Komagata O, Kasai S, Ogawa K, Tomita T - Sci Rep (2016)

Bottom Line: Here, we tested two genome editing technologies, transcription activator-like effector nucleases (TALEN)s and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), to disrupt CYP9M10 in a resistant strain of C. quinquefasciatus.Additionally, we developed a novel, effective approach to construct a TALE using the chemical cleavage of phosphorothioate inter-nucleotide linkages in the level 1 assembly.A line fixed with a completely disrupted CYP9M10 haplotype showed more than 100-fold reduction in pyrethroid resistance in the larval stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

ABSTRACT
Recently-emerging genome editing technologies have enabled targeted gene knockout experiments even in non-model insect species. For studies on insecticide resistance, genome editing technologies offer some advantages over the conventional reverse genetic technique, RNA interference, for testing causal relationships between genes of detoxifying enzymes and resistance phenotypes. There were relatively abundant evidences indicating that the overexpression of a cytochrome P450 gene CYP9M10 confers strong pyrethroid resistance in larvae of the southern house mosquito Culex quinquefasciatus. However, reverse genetic verification has not yet been obtained because of the technical difficulty of microinjection into larvae. Here, we tested two genome editing technologies, transcription activator-like effector nucleases (TALEN)s and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), to disrupt CYP9M10 in a resistant strain of C. quinquefasciatus. Additionally, we developed a novel, effective approach to construct a TALE using the chemical cleavage of phosphorothioate inter-nucleotide linkages in the level 1 assembly. Both TALEN and CRISPR/Cas9 induced frame-shifting mutations in one or all copies of CYP9M10 in a pyrethroid-resistant strain. A line fixed with a completely disrupted CYP9M10 haplotype showed more than 100-fold reduction in pyrethroid resistance in the larval stage.

No MeSH data available.


Related in: MedlinePlus

Permethrin susceptibility.The results of permethrin susceptibility assay are plotted. For #10, the assay was conducted at two different generations. The lines show the cumulative distribution function of a normal distribution function regressed on the result by the probit method.
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f4: Permethrin susceptibility.The results of permethrin susceptibility assay are plotted. For #10, the assay was conducted at two different generations. The lines show the cumulative distribution function of a normal distribution function regressed on the result by the probit method.

Mentions: Permethrin (one of pyrethroids insecticide) susceptibility was measured in fourth-instar larvae and compared to that of the parental JPP strain and pyrethroid-susceptible JNA strain (Fig. 4). The KO lines (M04, M07, 4–4 and 4–8) with one of the three (or two) copies disrupted showed little difference in susceptibility compared to the WT JPP strain. In contrast, #10, which carried no functional CYP9M10 copy showed an approximately 110-fold reduction in permethrin susceptibility from that of the WT JPP. The susceptibility was not recovered completely to the susceptible level (the JNA strain) because the JPP strain also carries a kdr mutation (L1014F) in the voltage-gated sodium channel gene3435, which is another genetic factor conferring pyrethroid resistance.


Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies.

Itokawa K, Komagata O, Kasai S, Ogawa K, Tomita T - Sci Rep (2016)

Permethrin susceptibility.The results of permethrin susceptibility assay are plotted. For #10, the assay was conducted at two different generations. The lines show the cumulative distribution function of a normal distribution function regressed on the result by the probit method.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837413&req=5

f4: Permethrin susceptibility.The results of permethrin susceptibility assay are plotted. For #10, the assay was conducted at two different generations. The lines show the cumulative distribution function of a normal distribution function regressed on the result by the probit method.
Mentions: Permethrin (one of pyrethroids insecticide) susceptibility was measured in fourth-instar larvae and compared to that of the parental JPP strain and pyrethroid-susceptible JNA strain (Fig. 4). The KO lines (M04, M07, 4–4 and 4–8) with one of the three (or two) copies disrupted showed little difference in susceptibility compared to the WT JPP strain. In contrast, #10, which carried no functional CYP9M10 copy showed an approximately 110-fold reduction in permethrin susceptibility from that of the WT JPP. The susceptibility was not recovered completely to the susceptible level (the JNA strain) because the JPP strain also carries a kdr mutation (L1014F) in the voltage-gated sodium channel gene3435, which is another genetic factor conferring pyrethroid resistance.

Bottom Line: Here, we tested two genome editing technologies, transcription activator-like effector nucleases (TALEN)s and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), to disrupt CYP9M10 in a resistant strain of C. quinquefasciatus.Additionally, we developed a novel, effective approach to construct a TALE using the chemical cleavage of phosphorothioate inter-nucleotide linkages in the level 1 assembly.A line fixed with a completely disrupted CYP9M10 haplotype showed more than 100-fold reduction in pyrethroid resistance in the larval stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

ABSTRACT
Recently-emerging genome editing technologies have enabled targeted gene knockout experiments even in non-model insect species. For studies on insecticide resistance, genome editing technologies offer some advantages over the conventional reverse genetic technique, RNA interference, for testing causal relationships between genes of detoxifying enzymes and resistance phenotypes. There were relatively abundant evidences indicating that the overexpression of a cytochrome P450 gene CYP9M10 confers strong pyrethroid resistance in larvae of the southern house mosquito Culex quinquefasciatus. However, reverse genetic verification has not yet been obtained because of the technical difficulty of microinjection into larvae. Here, we tested two genome editing technologies, transcription activator-like effector nucleases (TALEN)s and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), to disrupt CYP9M10 in a resistant strain of C. quinquefasciatus. Additionally, we developed a novel, effective approach to construct a TALE using the chemical cleavage of phosphorothioate inter-nucleotide linkages in the level 1 assembly. Both TALEN and CRISPR/Cas9 induced frame-shifting mutations in one or all copies of CYP9M10 in a pyrethroid-resistant strain. A line fixed with a completely disrupted CYP9M10 haplotype showed more than 100-fold reduction in pyrethroid resistance in the larval stage.

No MeSH data available.


Related in: MedlinePlus