Zinc finger nuclease‐mediated precision genome editing of an endogenous gene in hexaploid bread wheat (Triticum aestivum) using a DNA repair template

Abstract

SummarySequence‐specific nucleases have been used to engineer targeted genome modifications in various plants. While targeted gene knockouts resulting in loss of function have been reported with relatively high rates of success, targeted gene editing using an exogenously supplied DNA repair template and site‐specific transgene integration has been more challenging. Here, we report the first application of zinc finger nuclease (ZFN)‐mediated, nonhomologous end‐joining (NHEJ)‐directed editing of a native gene in allohexaploid bread wheat to introduce, via a supplied DNA repair template, a specific single amino acid change into the coding sequence of acetohydroxyacid synthase (AHAS) to confer resistance to imidazolinone herbicides. We recovered edited wheat plants having the targeted amino acid modification in one or more AHAS homoalleles via direct selection for resistance to imazamox, an AHAS‐inhibiting imidazolinone herbicide. Using a cotransformation strategy based on chemical selection for an exogenous marker, we achieved a 1.2% recovery rate of edited plants having the desired amino acid change and a 2.9% recovery of plants with targeted mutations at the AHAS locus resulting in a loss‐of‐function gene knockout. The latter results demonstrate a broadly applicable approach to introduce targeted modifications into native genes for nonselectable traits. All ZFN‐mediated changes were faithfully transmitted to the next generation.

A reductionist approach to dissecting grain weight and yield in wheat

“…In the future, understanding the relationship between multiple genes affecting yield components will most likely require multiplex genome editing of the different targets across several genetic backgrounds (Shen et al ). This will involve not only knock‐out mutations but also allele replacement and promoter manipulations (Puchta ; Rodríguez‐Leal et al ; Li et al ; Ran et al ).…”

Genome Editing in Agriculture: Technical and Practical Considerations

“…Both circular and linear DNA can be introduced into plant cells by particle bombardment, and the co-delivery of different DNA fragments or plasmids is quite efficient because they can be coated onto the same microparticles [121,122]. A recent study in wheat demonstrated the ZFN-mediated precise editing of the three homoeologous ALS genes following bombardment with the ZFN construct and a short dsDNA donor with compatible overhangs [76].…”

“…TALENs and the CRISPR/Cas9 system have been used for nucleotide exchange in the rice gene encoding acetolactate synthase ( ALS ), generating plants resistant to bispyribac-sodium [75]. Furthermore, ZFNs have been used for nucleotide exchange in the wheat ALS gene to confer resistance to imidazolinone herbicides [76]. By delivering the CRISPR/Cas9 components either as DNA or RNP in combination with single-strand oligonucleotides, the maize ALS2 gene was also edited and chlorsulfuron-resistant plants were obtained [77,78].…”

Cas Endonuclease Technology—A Quantum Leap in the Advancement of Barley and Wheat Genetic Engineering

“…In this case, a strain of the Bean Yellow Dwarf Virus was modified so that only the elements essential for the method remained and the artificial repair template was amplified with high frequency [110]. In barley and wheat, precise editing has been achieved either only at the cellular level [98] or has the limiting prerequisite that the obtained genetic modification leads to an in vitro selectable trait, e.g., an herbicide resistance [111].…”