Ayako Nishizawa-Yokoi, Masaki Endo, Namie Ohtsuki, Hiroaki Saika, Seiichi Toki

Plant Genome Engineering Research Unit

［Abstract］

We have succeeded in establishing a technique that allows pinpoint genome editing in rice. This is made possible by gene targeting with positive-negative selection and subsequent marker excision via piggyBac transposon.

［Keywords］

Keywords: gene targeting, positive-negative selection, marker excision, piggyBac transposon

［Background］

Gene targeting (GT) is a technique for accurate genome editing. Using GT with positive-negative selection, the positive selection marker gene should be removed completely from GT locus. However, no such system has been established so far. Here, we have succeeded in accurate excision of marker without leaving a footprint at the excise site using insect-derived piggyBac transposon.

［Results and Discussion］

1. We attempted to introduce targeted two point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with positive-negative selection (Fig. 1A:Step1). Subsequently, the positive marker gene was excised by piggyBac transposition leaving targeted two point mutations on the ALS gene (Fig. 1A:Step2).
   
2. The transgenic calli harboring a modified ALS locus containing point mutations and positive selection marker were then selected and subjected to Step2. PCR analysis revealed that the marker gene was excised completely from ALS locus by piggyBac transposition in 99 out of 100 regenerated plants from five independent calli lines (Fig. 1B). Sequencing analysis demonstrated transposition of piggyBac without leaving a footprint at the excise site.
   
3. We confirmed that the transcripts of modified ALS locus were comparable to that of wild-type ALS gene in T₁ progenies obtained from marker-free regenerated plants (Fig. 2A and 2B). Furthermore, T₁ progenies containing modified ALS locus showed the herbicide-tolerant phenotype (Fig. 2C).
   
4. This approach was also applied successfully in pinpoint mutagenesis of several genes in rice.

1. The marker-free T₁ progenies containing the two point mutations in ALS gene but without the expression cassette of piggyBac transposase were obtained from marker-free regenerated plants. These plants contained only the targeted point mutations in the ALS gene without any dispensable sequences and are therefore similar to plants generated by classical mutation breeding techniques.
   
2. The piggyBac mediated transposition system could be used not only in rice but also in other plant species. The development of an effective GT system with positive-negative selection could facilitate pinpoint mutagenesis of target gene in several plant species.

Fig. 1. Strategy for the introduction of point mutations into the ALS locus via GT and subsequent marker excision from GT locus using piggyBac transposon. (A) Schematic diagram of GT at the ALS locus. Step1:The point mutations conferring resistance to herbicide (red star) and positive selection marker were introduced into the ALS locus by homologous recombination. Step2:GT calli were again infected with Agrobacterium harboring a transposase (PBase) expression vector and the positive selection marker was excised by piggyBac transposition. (B) The frequency of piggyBac-mediated marker excision in PBase-expressing regenerated plants.

 

Fig. 2. Analysis of the ALS gene harboring mutations in T1 progenies. (A) Diagram showing the targeted ALS locus. (B) CAPS analysis combining PCR analysis using ALS gene-specific primers (arrowheads) with cDNA and MfeI digestion in T1 plants carrying the wild-type (wt) or modified ALS gene (heterozygous or homozygous). (C) Herbicide-tolerant phenotype of T1 plants.

 

[Reference]

1. Nishizawa-Yokoi A, Endo M, Ohtsuki N, Saika H, Toki S (2015) Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision The Plant Journal 81 (1):160-168