Limits...
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

Sequence pherograms of each fixed KO line.(A) The v1 copy and v2 copies of CYP9M10 were directly sequenced for each fixed KO line. The horizontal arrowheads indicate primers used to amplify each fragment as colours corresponding to Fig. 2A. (B) Showing a model for three CYP9M10 copies (two v2 copies).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4837413&req=5

f3: Sequence pherograms of each fixed KO line.(A) The v1 copy and v2 copies of CYP9M10 were directly sequenced for each fixed KO line. The horizontal arrowheads indicate primers used to amplify each fragment as colours corresponding to Fig. 2A. (B) Showing a model for three CYP9M10 copies (two v2 copies).

Mentions: The genotype frequencies at the IBC1 generation in the 4–4 line during the fixation process (Fig. 2B) departed significantly from the expected ratio (1:2:1) of the normal Mendelian inheritance model (Table 2 and Fig. S2), suggesting that the knocked-out CYP9M10 haplotype was linked to lower survival in this line. Since the other lines did not show such a large departure from the Mendelian model, it is not clear whether the lower survival in the 4–4 line was due to the knockout of CYP9M10. Several generations after establishment of KO lines, the v1 and v2 copies were separately amplified and sequenced directly for four individuals in each line. All four individuals in the M07 and 4–4 lines each carrying the disrupted v1 copy showed sequence pherograms with homozygous peaks for both v1 and v2 copies. In contrast, all four individuals in the M40 and 4–8 lines each carrying a disrupted v2 copy still showed heterozygous poly peaks composed of the mutated and WT alleles (Fig. 3A) in the v2 fragment. We checked another 16 individuals (eight females and eight males) from each M40 and 4–8 line after further several inbreeding generations and confirmed all individuals still showed same heterozygous sequence pherograms for v2 fragment as seen in Fig. 3A. Thus, the heterogeneity was not due to incomplete fixation (p < 0.0004 by the chi-square test for Hardy–Weinberg equilibrium) but rather indicating the presence of two v2 copies (thus, three copies of CYP9M10 in total) per one haplotype, at least in those lines (Fig. 3A), with only one of the two having been mutated. Our previous estimate of the CYP9M10 copy number in the JPP strain was two1619. This discrepancy was probably simply due to the technical difficulty of distinguishing between two and three copies (a 1.5-fold difference) by quantitative PCR-based methods. Another, but non-mutually exclusive, hypothesis is that the copy numbers are polymorphic within the JPP strain because of instability of tandem duplication due to unequal crossing-over or replication slippage. Targeting two sites in the same chromosome concurrently may also induce a de novo duplication event by inter-chromosomal or -chromatid ligation32. Both the v1 and v2 fragments amplified from the four individuals in #10 line created by CRISPR/Cas9, on the other hand, showed homozygous peaks for the mutated alleles (Fig. 3B). At present we cannot determine the precise copy number status in the original JPP strain. Nevertheless, at least the M40 and 4–8 lines still carried two intact copies of CYP9M10 and the #10 line was considered to no longer harbor a functional CYP9M10 copy.


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)

Sequence pherograms of each fixed KO line.(A) The v1 copy and v2 copies of CYP9M10 were directly sequenced for each fixed KO line. The horizontal arrowheads indicate primers used to amplify each fragment as colours corresponding to Fig. 2A. (B) Showing a model for three CYP9M10 copies (two v2 copies).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Sequence pherograms of each fixed KO line.(A) The v1 copy and v2 copies of CYP9M10 were directly sequenced for each fixed KO line. The horizontal arrowheads indicate primers used to amplify each fragment as colours corresponding to Fig. 2A. (B) Showing a model for three CYP9M10 copies (two v2 copies).
Mentions: The genotype frequencies at the IBC1 generation in the 4–4 line during the fixation process (Fig. 2B) departed significantly from the expected ratio (1:2:1) of the normal Mendelian inheritance model (Table 2 and Fig. S2), suggesting that the knocked-out CYP9M10 haplotype was linked to lower survival in this line. Since the other lines did not show such a large departure from the Mendelian model, it is not clear whether the lower survival in the 4–4 line was due to the knockout of CYP9M10. Several generations after establishment of KO lines, the v1 and v2 copies were separately amplified and sequenced directly for four individuals in each line. All four individuals in the M07 and 4–4 lines each carrying the disrupted v1 copy showed sequence pherograms with homozygous peaks for both v1 and v2 copies. In contrast, all four individuals in the M40 and 4–8 lines each carrying a disrupted v2 copy still showed heterozygous poly peaks composed of the mutated and WT alleles (Fig. 3A) in the v2 fragment. We checked another 16 individuals (eight females and eight males) from each M40 and 4–8 line after further several inbreeding generations and confirmed all individuals still showed same heterozygous sequence pherograms for v2 fragment as seen in Fig. 3A. Thus, the heterogeneity was not due to incomplete fixation (p < 0.0004 by the chi-square test for Hardy–Weinberg equilibrium) but rather indicating the presence of two v2 copies (thus, three copies of CYP9M10 in total) per one haplotype, at least in those lines (Fig. 3A), with only one of the two having been mutated. Our previous estimate of the CYP9M10 copy number in the JPP strain was two1619. This discrepancy was probably simply due to the technical difficulty of distinguishing between two and three copies (a 1.5-fold difference) by quantitative PCR-based methods. Another, but non-mutually exclusive, hypothesis is that the copy numbers are polymorphic within the JPP strain because of instability of tandem duplication due to unequal crossing-over or replication slippage. Targeting two sites in the same chromosome concurrently may also induce a de novo duplication event by inter-chromosomal or -chromatid ligation32. Both the v1 and v2 fragments amplified from the four individuals in #10 line created by CRISPR/Cas9, on the other hand, showed homozygous peaks for the mutated alleles (Fig. 3B). At present we cannot determine the precise copy number status in the original JPP strain. Nevertheless, at least the M40 and 4–8 lines still carried two intact copies of CYP9M10 and the #10 line was considered to no longer harbor a functional CYP9M10 copy.

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