Precise genetic engineering with piggyBac transposon in plants

Abstract

Transposons are mobile genetic elements that can move to a different position within a genome or between genomes. They have long been used as a tool for genetic engineering, including transgenesis, insertional mutagenesis, and marker excision, in a variety of organisms. The piggyBac transposon derived from the cabbage looper moth is one of the most promising transposon tools ever identified because piggyBac has the advantage that it can transpose without leaving a footprint at the excised site. Applying the piggyBac transposon to precise genome editing in plants, we have demonstrated efficient and precise piggyBac transposon excision from a transgene locus integrated into the rice genome. Furthermore, introduction of only desired point mutations into the target gene can be achieved by a combination of precise gene modification via homologous recombination-mediated gene targeting with subsequent marker excision from target loci using piggyBac transposition in rice. In addition, we have designed a piggyBac-mediated transgenesis system for the temporary expression of sequence-specific nucleases to eliminate the transgene from the host genome without leaving unnecessary sequences after the successful induction of targeted mutagenesis via sequence-specific nucleases for use in vegetatively propagated plants. In this review, we summarize our previous works and the future prospects of genetic engineering with piggyBac transposon.

Keywords: CRISPR/Cas9; DNA double-strand breaks; homologous recombination-mediated gene targeting; piggyBac transposon; rice.

Figure 1. Schematic representation of the transposition reaction of piggyBac transposon. A, Diagram of piggyBac transposon. ITR, inverted terminal repeat; IR, sub-terminal inverted repeat. The piggyBac transposon is excised from the host genome by the expression of piggyBac transposase (PBase). B, Scheme of piggyBac transposition. PBase recognizes the terminal repeats and catalyzes transposition and integration into the TTAA element in the genome. The TTAA element is duplicated along the edges of inverted repeats of piggyBac during the integration of this transposon and returns to a single TTAA element in the subsequent excision step.

Figure 2. Efficient and precise transposition of piggyBac in PBase-expressing rice calli. A, Schematic representation of reporter constructs used to detect piggyBac transposition as luciferase luminescence in rice calli. Upon precise transposition of piggyBac from the luciferase (LUC) gene, transgenic calli become LUC-positive. B, C, Images of rice calli expressing GFP as a control (B) and hyPBase (C).

Figure 3. Strategy to introduce desired modifications into a gene of interest (GOI) via GT with positive-negative selection and subsequent marker excision from the GOI locus using piggyBac transposon. Step 1, Introduction of desired modifications (star) into the target gene via GT with positive-negative selection. The GT vector comprises the homologous sequence of target gene locus (white boxes and thick lines) with desired modifications (star), the piggyBac transposon carrying the positive selection marker (gray box), and the negative selection marker (black boxes). GT cells are enriched by positive-negative selection and are identified by PCR analysis. Step 2, Marker excision from the modified gene locus using piggyBac transposon. The expression cassette of hyPBase is transformed into GT calli to excise the positive selection marker via the transposition of piggyBac. If the piggyBac transposes without re-integration into another locus, the desired modifications are left behind in the target gene.

Figure 4. A strategy for piggyBac-mediated temporary transgenesis for the induction of targeted mutagenesis via CRISPR/Cas9 in plants. Step 1, The CRISPR/Cas9 expression cassette is integrated into the host genome from extrachromosomal T-DNA by piggyBac transposition, not by T-DNA integration, resulting from transient expression of hyPBase on T-DNA. Step 2, DNA double-strand breaks (DSB) and DSB-derived mutations are induced at the target gene by expression of CRISPR/Cas9. Step 3, piggyBac transposon is excised precisely from the host genome by expression of hyPBase, leaving only the targeted mutations in the target gene.