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Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster.

Simoni A, Siniscalchi C, Chan YS, Huen DS, Russell S, Windbichler N, Crisanti A - Nucleic Acids Res. (2014)

Bottom Line: It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications.We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as 'homing' similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage.We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.

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Schematic representation of the genetic markers used to follow the structure of the progeny. (A) Donor and target constructs were inserted by ΦC31site-specific integration in the same attP2 docking line (on chromosome 3L, position 11 063 638 bp). Donor nuclease sequences are inserted as cassette (top) within their corresponding target site interrupting the green fluorescent protein (GFP) coding sequence. Three different SSEs were constructed encompassing the following elements: the male germline promoter Rcd-1r (white triangle), a TALEN or a ZFN-pair nuclease (blue shape) and the ß56D-tubulin 3’-UTR (black bar). The TALELAT construct carries a RFP marker gene driven by the eye-specific promoter 3xP3 (black triangle). The left and the right ZFNs are separated by a Furin-2A self-cleavage ribosomal stuttering peptide (yellow). The donor constructs are adjacent to a functional mini-white gene (orange box) that restores the red pigmentation in the fly's eyes as phenotypical marker and the recessive marker curled (cu), on chromosome 3R (position 7 023 314). The recipient (target) chromosome carries the nuclease target sequence (as shown. The nuclease recognition sequences are underlined and in capital letters) in-frame with a functional GFP gene (green box), driven by the eye-specific promoter 3xP3. The mini-white marker in the target line was inactivated by a frame-shift mutation (marked by an arrow head). (B) Schematic of the homing assay. Donor/target trans-heterozygous flies are crossed to attP2 cu flies (the genetic background). When the nucleases are expressed in the germline, the target site is cleaved and the chromosome repair mechanisms can lead to different progeny outcomes, which can be discriminated by fluorescent and phenotypical markers, as indicated. Donor and target chromosome (marked in grey and white, respectively) can be discriminated by the presence of dominant mini-white (w) and recessive curled (cu) markers. We defined ‘homing’ as any recombination event which leads to the conversion of the target chromosome into a nuclease expressing chromosome. (C) PCR reactions were performed on w- GFP-negative flies with different sets of primers to distinguish HR-dependent repair events from imprecise NHEJ. The combination P1–P2 generates a PCR amplicon only in the case of homing of the locus while the combination P1–P3 is diagnostic for NHEJ events. Lanes 1–10 were loaded with PCR reactions generated from GFP-negative flies derived from TH males crossed to wt females.
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Figure 2: Schematic representation of the genetic markers used to follow the structure of the progeny. (A) Donor and target constructs were inserted by ΦC31site-specific integration in the same attP2 docking line (on chromosome 3L, position 11 063 638 bp). Donor nuclease sequences are inserted as cassette (top) within their corresponding target site interrupting the green fluorescent protein (GFP) coding sequence. Three different SSEs were constructed encompassing the following elements: the male germline promoter Rcd-1r (white triangle), a TALEN or a ZFN-pair nuclease (blue shape) and the ß56D-tubulin 3’-UTR (black bar). The TALELAT construct carries a RFP marker gene driven by the eye-specific promoter 3xP3 (black triangle). The left and the right ZFNs are separated by a Furin-2A self-cleavage ribosomal stuttering peptide (yellow). The donor constructs are adjacent to a functional mini-white gene (orange box) that restores the red pigmentation in the fly's eyes as phenotypical marker and the recessive marker curled (cu), on chromosome 3R (position 7 023 314). The recipient (target) chromosome carries the nuclease target sequence (as shown. The nuclease recognition sequences are underlined and in capital letters) in-frame with a functional GFP gene (green box), driven by the eye-specific promoter 3xP3. The mini-white marker in the target line was inactivated by a frame-shift mutation (marked by an arrow head). (B) Schematic of the homing assay. Donor/target trans-heterozygous flies are crossed to attP2 cu flies (the genetic background). When the nucleases are expressed in the germline, the target site is cleaved and the chromosome repair mechanisms can lead to different progeny outcomes, which can be discriminated by fluorescent and phenotypical markers, as indicated. Donor and target chromosome (marked in grey and white, respectively) can be discriminated by the presence of dominant mini-white (w) and recessive curled (cu) markers. We defined ‘homing’ as any recombination event which leads to the conversion of the target chromosome into a nuclease expressing chromosome. (C) PCR reactions were performed on w- GFP-negative flies with different sets of primers to distinguish HR-dependent repair events from imprecise NHEJ. The combination P1–P2 generates a PCR amplicon only in the case of homing of the locus while the combination P1–P3 is diagnostic for NHEJ events. Lanes 1–10 were loaded with PCR reactions generated from GFP-negative flies derived from TH males crossed to wt females.

Mentions: Transgenic fly lines were produced by ϕC31 integrase-mediated insertion into the attP2 docking line (3L: 11 063 638). The TALELAT donor and AAVS1 target were inserted into an otherwise unmarked chromosome. ZFN-AAVS1, ZFN-AAVS1-Long donor and TALELAT target were inserted into an attP2 chromosome marked with curled (cu). The schematic of the homing assay and the possible progeny outcomes from the trans-heterozygous cross depicted in Figure 2B and described in the text in reference to the curled marker exemplifies the situation of the TALELAT line. For ZFN-AAVS1 and ZFN-AAVS1-Long, the curled marker is on the target chromosome, and the progeny was classified accordingly.


Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster.

Simoni A, Siniscalchi C, Chan YS, Huen DS, Russell S, Windbichler N, Crisanti A - Nucleic Acids Res. (2014)

Schematic representation of the genetic markers used to follow the structure of the progeny. (A) Donor and target constructs were inserted by ΦC31site-specific integration in the same attP2 docking line (on chromosome 3L, position 11 063 638 bp). Donor nuclease sequences are inserted as cassette (top) within their corresponding target site interrupting the green fluorescent protein (GFP) coding sequence. Three different SSEs were constructed encompassing the following elements: the male germline promoter Rcd-1r (white triangle), a TALEN or a ZFN-pair nuclease (blue shape) and the ß56D-tubulin 3’-UTR (black bar). The TALELAT construct carries a RFP marker gene driven by the eye-specific promoter 3xP3 (black triangle). The left and the right ZFNs are separated by a Furin-2A self-cleavage ribosomal stuttering peptide (yellow). The donor constructs are adjacent to a functional mini-white gene (orange box) that restores the red pigmentation in the fly's eyes as phenotypical marker and the recessive marker curled (cu), on chromosome 3R (position 7 023 314). The recipient (target) chromosome carries the nuclease target sequence (as shown. The nuclease recognition sequences are underlined and in capital letters) in-frame with a functional GFP gene (green box), driven by the eye-specific promoter 3xP3. The mini-white marker in the target line was inactivated by a frame-shift mutation (marked by an arrow head). (B) Schematic of the homing assay. Donor/target trans-heterozygous flies are crossed to attP2 cu flies (the genetic background). When the nucleases are expressed in the germline, the target site is cleaved and the chromosome repair mechanisms can lead to different progeny outcomes, which can be discriminated by fluorescent and phenotypical markers, as indicated. Donor and target chromosome (marked in grey and white, respectively) can be discriminated by the presence of dominant mini-white (w) and recessive curled (cu) markers. We defined ‘homing’ as any recombination event which leads to the conversion of the target chromosome into a nuclease expressing chromosome. (C) PCR reactions were performed on w- GFP-negative flies with different sets of primers to distinguish HR-dependent repair events from imprecise NHEJ. The combination P1–P2 generates a PCR amplicon only in the case of homing of the locus while the combination P1–P3 is diagnostic for NHEJ events. Lanes 1–10 were loaded with PCR reactions generated from GFP-negative flies derived from TH males crossed to wt females.
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Figure 2: Schematic representation of the genetic markers used to follow the structure of the progeny. (A) Donor and target constructs were inserted by ΦC31site-specific integration in the same attP2 docking line (on chromosome 3L, position 11 063 638 bp). Donor nuclease sequences are inserted as cassette (top) within their corresponding target site interrupting the green fluorescent protein (GFP) coding sequence. Three different SSEs were constructed encompassing the following elements: the male germline promoter Rcd-1r (white triangle), a TALEN or a ZFN-pair nuclease (blue shape) and the ß56D-tubulin 3’-UTR (black bar). The TALELAT construct carries a RFP marker gene driven by the eye-specific promoter 3xP3 (black triangle). The left and the right ZFNs are separated by a Furin-2A self-cleavage ribosomal stuttering peptide (yellow). The donor constructs are adjacent to a functional mini-white gene (orange box) that restores the red pigmentation in the fly's eyes as phenotypical marker and the recessive marker curled (cu), on chromosome 3R (position 7 023 314). The recipient (target) chromosome carries the nuclease target sequence (as shown. The nuclease recognition sequences are underlined and in capital letters) in-frame with a functional GFP gene (green box), driven by the eye-specific promoter 3xP3. The mini-white marker in the target line was inactivated by a frame-shift mutation (marked by an arrow head). (B) Schematic of the homing assay. Donor/target trans-heterozygous flies are crossed to attP2 cu flies (the genetic background). When the nucleases are expressed in the germline, the target site is cleaved and the chromosome repair mechanisms can lead to different progeny outcomes, which can be discriminated by fluorescent and phenotypical markers, as indicated. Donor and target chromosome (marked in grey and white, respectively) can be discriminated by the presence of dominant mini-white (w) and recessive curled (cu) markers. We defined ‘homing’ as any recombination event which leads to the conversion of the target chromosome into a nuclease expressing chromosome. (C) PCR reactions were performed on w- GFP-negative flies with different sets of primers to distinguish HR-dependent repair events from imprecise NHEJ. The combination P1–P2 generates a PCR amplicon only in the case of homing of the locus while the combination P1–P3 is diagnostic for NHEJ events. Lanes 1–10 were loaded with PCR reactions generated from GFP-negative flies derived from TH males crossed to wt females.
Mentions: Transgenic fly lines were produced by ϕC31 integrase-mediated insertion into the attP2 docking line (3L: 11 063 638). The TALELAT donor and AAVS1 target were inserted into an otherwise unmarked chromosome. ZFN-AAVS1, ZFN-AAVS1-Long donor and TALELAT target were inserted into an attP2 chromosome marked with curled (cu). The schematic of the homing assay and the possible progeny outcomes from the trans-heterozygous cross depicted in Figure 2B and described in the text in reference to the curled marker exemplifies the situation of the TALELAT line. For ZFN-AAVS1 and ZFN-AAVS1-Long, the curled marker is on the target chromosome, and the progeny was classified accordingly.

Bottom Line: It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications.We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as 'homing' similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage.We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.

Show MeSH
Related in: MedlinePlus