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Zinc finger nuclease-mediated transgene deletion

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Abstract

A transgene, flanked by zinc finger nuclease (ZFN) cleavage sites, was deleted from a stably transformed plant by crossing it with a second plant expressing a corresponding ZFN gene. A target construct, containing a GUS reporter gene flanked by ZFN cleavage sites, a GFP reporter gene and a PAT selectable marker gene, was transformed into tobacco. Basta®-resistant plants were regenerated and screened for GUS and GFP expression. A second construct, containing a ZFN gene driven by the constitutive CsVMV promoter and an HPT selectable marker gene, was also transformed into tobacco. Selected T0 plants were grown to maturity and allowed to self-pollinate. Homozygous target plants, which expressed GUS and GFP, were crossed with homozygous ZFN plants, which expressed the ZFN gene. Numerous GUS-negative plants were observed among the hybrids with one particular cross displaying ~35% GUS-negative plants. Evidence for complete deletion of a 4.3 kb sequence comprising the GUS gene was obtained and sequence confirmed. Co-segregation in F2 progenies of ‘truncated’ and ‘intact’ target sequences with expected reporter gene phenotypes were observed. Since ZFNs can be designed to bind and cleave a wide range of DNA sequences, these results constitute a general strategy for creating targeted gene deletions.

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Acknowledgments

Thanks to Fyodor Urnov and the staff at Sangamo BioSciences for providing the CCR5 binding sites and corresponsing ZFN and to Rebekah Bark, Sushma Ram and Greg Schulenberg for assistance with progeny GUS analysis, qRT-PCR and Southern blots/PCR, respectively.

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Correspondence to Joseph F. Petolino.

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Petolino, J.F., Worden, A., Curlee, K. et al. Zinc finger nuclease-mediated transgene deletion. Plant Mol Biol 73, 617–628 (2010). https://doi.org/10.1007/s11103-010-9641-4

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