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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

PC assembly of TALE hexamer/pentamer.(A) Structure of 1.5-mer template is shown. Phosphorothioate modified primers used were depicted blow. The sequences of the primers are listed in Table S1 in the supplemental online file. (B) PCR amplified monomers are assembled into hexamer or pentamer. The names of fragments (I–VIII) correspond to Fig. 1A. Note that the fragments VII and VIII work as adjusters for pentamer and hexamer assemblies, respectively. Each construct possesses BsaI site at both ends which will be used for subsequent full length TALE construction by Golden Gate reaction. (C) Colony check PCR was conducted for clones of hexamer/pentamer for TALE9M10L & R assembled by PC. The PCR products were electrophoresed in a microchip electrophoresis system MCE-202 MultiNa (Shimadzu, Kyoto, Japan). “UM” and “LM” each indicate the upper and lower markers, respectively. Arrowheads on right side indicate expected fragment size for correctly assembled hexamers and pentamers.
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f1: PC assembly of TALE hexamer/pentamer.(A) Structure of 1.5-mer template is shown. Phosphorothioate modified primers used were depicted blow. The sequences of the primers are listed in Table S1 in the supplemental online file. (B) PCR amplified monomers are assembled into hexamer or pentamer. The names of fragments (I–VIII) correspond to Fig. 1A. Note that the fragments VII and VIII work as adjusters for pentamer and hexamer assemblies, respectively. Each construct possesses BsaI site at both ends which will be used for subsequent full length TALE construction by Golden Gate reaction. (C) Colony check PCR was conducted for clones of hexamer/pentamer for TALE9M10L & R assembled by PC. The PCR products were electrophoresed in a microchip electrophoresis system MCE-202 MultiNa (Shimadzu, Kyoto, Japan). “UM” and “LM” each indicate the upper and lower markers, respectively. Arrowheads on right side indicate expected fragment size for correctly assembled hexamers and pentamers.

Mentions: In this study, we disrupted a detoxification enzyme gene in a pest insect using TALENs and CRISPR to validate the causality of an insecticide resistance phenotype. We also developed a novel method to construct TALEs using phosphorothioate chemistry (PC)15 (Fig. 1). As a proof of concept, we targeted CYP9M10 in C. quinquefasciatus. CYP9M10 encodes a cytochrome P450 that degrades pyrethroid insecticides in vitro2. CYP9M10 is overexpressed in a strongly pyrethroid-resistant strain, JPal-per (JPP)3, and is linked to the resistance phenotype18. The upregulation of CYP9M10 in the JPP strain is due to multiple cis-acting mutations and recent gene duplication161718. Althouhg the two duplicated copies have the same sequence in the transcribed region, a junction formed by the tandem duplication of a 100-kb segment is located 1.1 kb upstream of the transcription start site of CYP9M10 and differentiates the two copies (v1 and v2)16. Although, much evidences support the causal relationship between CYP9M10 and pyrethroid resistance2161719, RNAi verification had not been conducted because the gene overexpression and resistance are specific to the larval stage, which is highly vulnerable to microinjection20. Both TALENs and gRNA targeting CYP9M10 introduced frame-shift mutations in the JPP strain. We obtained lines fixed with each haplotype mutated in either v1 or v2 copies of CYP9M10, or both. Dramatic reduction (110-fold) in permethrin (one of pyrethroid insecticides) resistance was observed in a line in which all CYP9M10 copies were disrupted.


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)

PC assembly of TALE hexamer/pentamer.(A) Structure of 1.5-mer template is shown. Phosphorothioate modified primers used were depicted blow. The sequences of the primers are listed in Table S1 in the supplemental online file. (B) PCR amplified monomers are assembled into hexamer or pentamer. The names of fragments (I–VIII) correspond to Fig. 1A. Note that the fragments VII and VIII work as adjusters for pentamer and hexamer assemblies, respectively. Each construct possesses BsaI site at both ends which will be used for subsequent full length TALE construction by Golden Gate reaction. (C) Colony check PCR was conducted for clones of hexamer/pentamer for TALE9M10L & R assembled by PC. The PCR products were electrophoresed in a microchip electrophoresis system MCE-202 MultiNa (Shimadzu, Kyoto, Japan). “UM” and “LM” each indicate the upper and lower markers, respectively. Arrowheads on right side indicate expected fragment size for correctly assembled hexamers and pentamers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: PC assembly of TALE hexamer/pentamer.(A) Structure of 1.5-mer template is shown. Phosphorothioate modified primers used were depicted blow. The sequences of the primers are listed in Table S1 in the supplemental online file. (B) PCR amplified monomers are assembled into hexamer or pentamer. The names of fragments (I–VIII) correspond to Fig. 1A. Note that the fragments VII and VIII work as adjusters for pentamer and hexamer assemblies, respectively. Each construct possesses BsaI site at both ends which will be used for subsequent full length TALE construction by Golden Gate reaction. (C) Colony check PCR was conducted for clones of hexamer/pentamer for TALE9M10L & R assembled by PC. The PCR products were electrophoresed in a microchip electrophoresis system MCE-202 MultiNa (Shimadzu, Kyoto, Japan). “UM” and “LM” each indicate the upper and lower markers, respectively. Arrowheads on right side indicate expected fragment size for correctly assembled hexamers and pentamers.
Mentions: In this study, we disrupted a detoxification enzyme gene in a pest insect using TALENs and CRISPR to validate the causality of an insecticide resistance phenotype. We also developed a novel method to construct TALEs using phosphorothioate chemistry (PC)15 (Fig. 1). As a proof of concept, we targeted CYP9M10 in C. quinquefasciatus. CYP9M10 encodes a cytochrome P450 that degrades pyrethroid insecticides in vitro2. CYP9M10 is overexpressed in a strongly pyrethroid-resistant strain, JPal-per (JPP)3, and is linked to the resistance phenotype18. The upregulation of CYP9M10 in the JPP strain is due to multiple cis-acting mutations and recent gene duplication161718. Althouhg the two duplicated copies have the same sequence in the transcribed region, a junction formed by the tandem duplication of a 100-kb segment is located 1.1 kb upstream of the transcription start site of CYP9M10 and differentiates the two copies (v1 and v2)16. Although, much evidences support the causal relationship between CYP9M10 and pyrethroid resistance2161719, RNAi verification had not been conducted because the gene overexpression and resistance are specific to the larval stage, which is highly vulnerable to microinjection20. Both TALENs and gRNA targeting CYP9M10 introduced frame-shift mutations in the JPP strain. We obtained lines fixed with each haplotype mutated in either v1 or v2 copies of CYP9M10, or both. Dramatic reduction (110-fold) in permethrin (one of pyrethroid insecticides) resistance was observed in a line in which all CYP9M10 copies were disrupted.

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