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Perspective on the combined use of an independent transgenic sexing and a multifactorial reproductive sterility system to avoid resistance development against transgenic Sterile Insect Technique approaches.

Eckermann KN, Dippel S, KaramiNejadRanjbar M, Ahmed HM, Curril IM, Wimmer EA - BMC Genet. (2014)

Bottom Line: However, to create 'redundant killing' an additional independent conditional expression system is required.We propose the combination of an already established transgenic embryonic sexing system to meet the SIT requirement of male-only releases based on the repressible tTA system together with a redundant male-specific reproductive sterility system, which is activated by Q-system controlled site-specific recombination and is based on a spermatogenesis-specifically expressed endonuclease acting on several species-specific target sites leading to chromosome shredding.A combination of a completely independent transgenic sexing and a redundant reproductive male sterility system, which do not share any active components and mediate the induced lethality by completely independent processes, would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs using biotechnologically enhanced SIT.

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ABSTRACT

Background: The Sterile Insect Technique (SIT) is an accepted species-specific genetic control approach that acts as an insect birth control measure, which can be improved by biotechnological engineering to facilitate its use and widen its applicability. First transgenic insects carrying a single killing system have already been released in small scale trials. However, to evade resistance development to such transgenic approaches, completely independent ways of transgenic killing should be established and combined.

Perspective: Most established transgenic sexing and reproductive sterility systems are based on the binary tTA expression system that can be suppressed by adding tetracycline to the food. However, to create 'redundant killing' an additional independent conditional expression system is required. Here we present a perspective on the use of a second food-controllable binary expression system - the inducible Q system - that could be used in combination with site-specific recombinases to generate independent transgenic killing systems. We propose the combination of an already established transgenic embryonic sexing system to meet the SIT requirement of male-only releases based on the repressible tTA system together with a redundant male-specific reproductive sterility system, which is activated by Q-system controlled site-specific recombination and is based on a spermatogenesis-specifically expressed endonuclease acting on several species-specific target sites leading to chromosome shredding.

Conclusion: A combination of a completely independent transgenic sexing and a redundant reproductive male sterility system, which do not share any active components and mediate the induced lethality by completely independent processes, would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs using biotechnologically enhanced SIT.

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Multifactorial reproductive sterility based on the CRISPR/Cas9 system causing chromosome shredding. The bacterial derived Cas9 endonuclease will be expressed under the control of the β2 tubulin (β2t) promoter. Cas9 will be targeted to para-centromeric, sub-telomeric, and diverse macrosatellite sequences by guide RNAs, which are encoded by a CRISPR RNA (crRNA) array. This crRNA array as well as the trans-acting crRNA (tracrRNA) will be expressed under diverse RNA polymerase III promoters such as from the snRNA U6 (U6:1, U6:3). In the crRNA array, the diverse crRNAs are separated by direct repeat sequences (DR) derived from the Streptococcus pyogenes CRISPR. The expressed Cas9 is loaded with tracrRNA and subsequently binds the crRNA array based on complementarity between tracrRNA and the DR sequences, thereby randomly selecting one of the crRNAs as a guide to produce a functional CRISPR/Cas9 endonuclease targeting the respective genomic loci [75], which will lead to double strand breaks causing chromosome shredding.
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Figure 3: Multifactorial reproductive sterility based on the CRISPR/Cas9 system causing chromosome shredding. The bacterial derived Cas9 endonuclease will be expressed under the control of the β2 tubulin (β2t) promoter. Cas9 will be targeted to para-centromeric, sub-telomeric, and diverse macrosatellite sequences by guide RNAs, which are encoded by a CRISPR RNA (crRNA) array. This crRNA array as well as the trans-acting crRNA (tracrRNA) will be expressed under diverse RNA polymerase III promoters such as from the snRNA U6 (U6:1, U6:3). In the crRNA array, the diverse crRNAs are separated by direct repeat sequences (DR) derived from the Streptococcus pyogenes CRISPR. The expressed Cas9 is loaded with tracrRNA and subsequently binds the crRNA array based on complementarity between tracrRNA and the DR sequences, thereby randomly selecting one of the crRNAs as a guide to produce a functional CRISPR/Cas9 endonuclease targeting the respective genomic loci [75], which will lead to double strand breaks causing chromosome shredding.

Mentions: By transgenic expression of several gRNAs using RNA polymerase III-dependent promoters, such as the U6 snRNA promoter, it has been shown that the Cas9 endonuclease can actually be targeted to several diverse targets, which can lead to a mutagenesis rate of up to 100% [71,72]. By our proposed use of the β2 tub promoter, Cas9 will be highly expressed during spermatogenesis and the mRNA still be highly translated during spermiogenesis [73] to expose the sperm chromosomes to high amounts of the endonuclease (Figure 3). To cause chromosome shredding, several guide RNAs can be employed to direct the CRISPR/Cas9 endonuclaese to para-centromeric, sub-telomeric, and microsatellite sequences. The induced double strand breaks will lead to large chromosomal aberrations causing aneuploidies that will mediate multifactorial reproductive sterility.


Perspective on the combined use of an independent transgenic sexing and a multifactorial reproductive sterility system to avoid resistance development against transgenic Sterile Insect Technique approaches.

Eckermann KN, Dippel S, KaramiNejadRanjbar M, Ahmed HM, Curril IM, Wimmer EA - BMC Genet. (2014)

Multifactorial reproductive sterility based on the CRISPR/Cas9 system causing chromosome shredding. The bacterial derived Cas9 endonuclease will be expressed under the control of the β2 tubulin (β2t) promoter. Cas9 will be targeted to para-centromeric, sub-telomeric, and diverse macrosatellite sequences by guide RNAs, which are encoded by a CRISPR RNA (crRNA) array. This crRNA array as well as the trans-acting crRNA (tracrRNA) will be expressed under diverse RNA polymerase III promoters such as from the snRNA U6 (U6:1, U6:3). In the crRNA array, the diverse crRNAs are separated by direct repeat sequences (DR) derived from the Streptococcus pyogenes CRISPR. The expressed Cas9 is loaded with tracrRNA and subsequently binds the crRNA array based on complementarity between tracrRNA and the DR sequences, thereby randomly selecting one of the crRNAs as a guide to produce a functional CRISPR/Cas9 endonuclease targeting the respective genomic loci [75], which will lead to double strand breaks causing chromosome shredding.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255789&req=5

Figure 3: Multifactorial reproductive sterility based on the CRISPR/Cas9 system causing chromosome shredding. The bacterial derived Cas9 endonuclease will be expressed under the control of the β2 tubulin (β2t) promoter. Cas9 will be targeted to para-centromeric, sub-telomeric, and diverse macrosatellite sequences by guide RNAs, which are encoded by a CRISPR RNA (crRNA) array. This crRNA array as well as the trans-acting crRNA (tracrRNA) will be expressed under diverse RNA polymerase III promoters such as from the snRNA U6 (U6:1, U6:3). In the crRNA array, the diverse crRNAs are separated by direct repeat sequences (DR) derived from the Streptococcus pyogenes CRISPR. The expressed Cas9 is loaded with tracrRNA and subsequently binds the crRNA array based on complementarity between tracrRNA and the DR sequences, thereby randomly selecting one of the crRNAs as a guide to produce a functional CRISPR/Cas9 endonuclease targeting the respective genomic loci [75], which will lead to double strand breaks causing chromosome shredding.
Mentions: By transgenic expression of several gRNAs using RNA polymerase III-dependent promoters, such as the U6 snRNA promoter, it has been shown that the Cas9 endonuclease can actually be targeted to several diverse targets, which can lead to a mutagenesis rate of up to 100% [71,72]. By our proposed use of the β2 tub promoter, Cas9 will be highly expressed during spermatogenesis and the mRNA still be highly translated during spermiogenesis [73] to expose the sperm chromosomes to high amounts of the endonuclease (Figure 3). To cause chromosome shredding, several guide RNAs can be employed to direct the CRISPR/Cas9 endonuclaese to para-centromeric, sub-telomeric, and microsatellite sequences. The induced double strand breaks will lead to large chromosomal aberrations causing aneuploidies that will mediate multifactorial reproductive sterility.

Bottom Line: However, to create 'redundant killing' an additional independent conditional expression system is required.We propose the combination of an already established transgenic embryonic sexing system to meet the SIT requirement of male-only releases based on the repressible tTA system together with a redundant male-specific reproductive sterility system, which is activated by Q-system controlled site-specific recombination and is based on a spermatogenesis-specifically expressed endonuclease acting on several species-specific target sites leading to chromosome shredding.A combination of a completely independent transgenic sexing and a redundant reproductive male sterility system, which do not share any active components and mediate the induced lethality by completely independent processes, would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs using biotechnologically enhanced SIT.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The Sterile Insect Technique (SIT) is an accepted species-specific genetic control approach that acts as an insect birth control measure, which can be improved by biotechnological engineering to facilitate its use and widen its applicability. First transgenic insects carrying a single killing system have already been released in small scale trials. However, to evade resistance development to such transgenic approaches, completely independent ways of transgenic killing should be established and combined.

Perspective: Most established transgenic sexing and reproductive sterility systems are based on the binary tTA expression system that can be suppressed by adding tetracycline to the food. However, to create 'redundant killing' an additional independent conditional expression system is required. Here we present a perspective on the use of a second food-controllable binary expression system - the inducible Q system - that could be used in combination with site-specific recombinases to generate independent transgenic killing systems. We propose the combination of an already established transgenic embryonic sexing system to meet the SIT requirement of male-only releases based on the repressible tTA system together with a redundant male-specific reproductive sterility system, which is activated by Q-system controlled site-specific recombination and is based on a spermatogenesis-specifically expressed endonuclease acting on several species-specific target sites leading to chromosome shredding.

Conclusion: A combination of a completely independent transgenic sexing and a redundant reproductive male sterility system, which do not share any active components and mediate the induced lethality by completely independent processes, would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs using biotechnologically enhanced SIT.

Show MeSH
Related in: MedlinePlus