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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

TALEN and CRISPR/Cas9 targets, fixation and fixed haplotypes.(A) Positions of TALEN9M10-3L & R and gRNA9M10-5 are shown as vertical arrowheads on translated region of CYP9M10. There are two CYP9M10 copies separated by approximately 100-kb in the JPP strain. “J” indicates the junction formed by the tandem duplication. Primers used for the SNiPerase assay and direct sequencing analysis were shown as horizontal arrowheads. (B) Crossing scheme to fix a mutant haplotype is diagrammed. “WT” indicates wild-type JPP counterpart for mating. “BC1” is backcross 1, and “IBC1” is inbred BC1. The box indicates genotyping method used in each step, as “HRMA” indicates high resolution melting analysis and “DS” indicate direct sequencing. (C) Haplotypes finally fixed in each knockout (KO) line. Bold characters indicate PAM for gRNA9M10-5.
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f2: TALEN and CRISPR/Cas9 targets, fixation and fixed haplotypes.(A) Positions of TALEN9M10-3L & R and gRNA9M10-5 are shown as vertical arrowheads on translated region of CYP9M10. There are two CYP9M10 copies separated by approximately 100-kb in the JPP strain. “J” indicates the junction formed by the tandem duplication. Primers used for the SNiPerase assay and direct sequencing analysis were shown as horizontal arrowheads. (B) Crossing scheme to fix a mutant haplotype is diagrammed. “WT” indicates wild-type JPP counterpart for mating. “BC1” is backcross 1, and “IBC1” is inbred BC1. The box indicates genotyping method used in each step, as “HRMA” indicates high resolution melting analysis and “DS” indicate direct sequencing. (C) Haplotypes finally fixed in each knockout (KO) line. Bold characters indicate PAM for gRNA9M10-5.

Mentions: Four TALE hexamers and two pentamers for TALEN9M10-3L & R (Fig. 2A) were assembled and cloned. As sizes of inserts were checked by colony PCR for 24 clones (four clones of each hexamer/pentamer), all except one (96%) hexamer clone showed single fragments with expected sizes (0.9 and 1.0 kb, respectively) (Fig. 1C). Among the 23 clones with inserts of correct-size, 22 (96%) had correct sequences. One hexamer clone contained a point mutation, which probably occurred during PCR. The correctly assembled clones of each hexamer/pentamer were used to assemble TALENs by Golden-Gate reaction using BsaI. The PCR amplified monomer fragments can be stored for at least six-months at −20 °C without an obvious decrease in cloning efficiency.


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)

TALEN and CRISPR/Cas9 targets, fixation and fixed haplotypes.(A) Positions of TALEN9M10-3L & R and gRNA9M10-5 are shown as vertical arrowheads on translated region of CYP9M10. There are two CYP9M10 copies separated by approximately 100-kb in the JPP strain. “J” indicates the junction formed by the tandem duplication. Primers used for the SNiPerase assay and direct sequencing analysis were shown as horizontal arrowheads. (B) Crossing scheme to fix a mutant haplotype is diagrammed. “WT” indicates wild-type JPP counterpart for mating. “BC1” is backcross 1, and “IBC1” is inbred BC1. The box indicates genotyping method used in each step, as “HRMA” indicates high resolution melting analysis and “DS” indicate direct sequencing. (C) Haplotypes finally fixed in each knockout (KO) line. Bold characters indicate PAM for gRNA9M10-5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: TALEN and CRISPR/Cas9 targets, fixation and fixed haplotypes.(A) Positions of TALEN9M10-3L & R and gRNA9M10-5 are shown as vertical arrowheads on translated region of CYP9M10. There are two CYP9M10 copies separated by approximately 100-kb in the JPP strain. “J” indicates the junction formed by the tandem duplication. Primers used for the SNiPerase assay and direct sequencing analysis were shown as horizontal arrowheads. (B) Crossing scheme to fix a mutant haplotype is diagrammed. “WT” indicates wild-type JPP counterpart for mating. “BC1” is backcross 1, and “IBC1” is inbred BC1. The box indicates genotyping method used in each step, as “HRMA” indicates high resolution melting analysis and “DS” indicate direct sequencing. (C) Haplotypes finally fixed in each knockout (KO) line. Bold characters indicate PAM for gRNA9M10-5.
Mentions: Four TALE hexamers and two pentamers for TALEN9M10-3L & R (Fig. 2A) were assembled and cloned. As sizes of inserts were checked by colony PCR for 24 clones (four clones of each hexamer/pentamer), all except one (96%) hexamer clone showed single fragments with expected sizes (0.9 and 1.0 kb, respectively) (Fig. 1C). Among the 23 clones with inserts of correct-size, 22 (96%) had correct sequences. One hexamer clone contained a point mutation, which probably occurred during PCR. The correctly assembled clones of each hexamer/pentamer were used to assemble TALENs by Golden-Gate reaction using BsaI. The PCR amplified monomer fragments can be stored for at least six-months at −20 °C without an obvious decrease in cloning efficiency.

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