Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision

Abstract

Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive-negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.

Keywords: Oryza sativa; acetolactate synthase; cleistogamy 1; gene targeting; marker excision; piggyBac transposon; technical advance.

Figure 1

Strategy for the introduction of point mutations into the ALS locus via GT and subsequent marker excision from the GT locus using piggyBac transposon. (a) Schematic diagram of GT at the ALS locus. The top line indicates the genomic structure of the wild-type ALS gene region. The bottom line shows the T-DNA region of the targeting vector carrying diphtheria toxin A subunit gene (DT-A) under the control of the maize polyubiquitin 1 promoter (P_ubi) or rice elongation factor-1α promoter (P_ef) as negative selection marker and a 6.4-kb fragment containing an ALS coding region (open box) with W548L and S627I mutations (red lines) and silent mutations (added HpaI site at 301-bp upstream of W548L; GCTGAC to GAATTC) for the insertion of piggyBac transposon (black triangle) harboring a rice actin terminator (T) and hpt gene under the control of the cauliflower mosaic virus 35S promoter (P_35S) as positive selection marker. LB, left border; RB, right border. (b) Strategy for precise marker excision using piggyBac transposon from the GT locus. The top line reveals the structure of the modified ALS locus resulting from homologous recombination between the targeting vector and wild-type locus. The bottom line represents the ALS locus modified by GT and subsequent precise marker excision via piggyBac transposition. The primer sets used for PCR that identify transgenic calli in which a GT event occurred at ALS locus are shown as black arrows. White arrows indicate the primer sets used for CAPS analysis to evaluate the frequency of marker excision via piggyBac transposition. Gray arrows represent primers for PCR analysis to detect the existence of re-integrated piggyBac transposon. The numbers on each arrow reveal the length of the PCR fragments. (c) Sequencing chromatograms of the excision site and mutation site in T₀ plants. (d–f) Southern blot analysis with probe1 (d), 2 (f), and 3 (e) shown in (a) and (b) using MfeI-digested genomic DNA of wild-type, a regenerated plant of ALS GT-A and two T₀ plants each of two independent lines with ALS GT-A_hy-6 and 10 (ALS GT-A_hy-6-1, 6-2, 10-1 and 10-2).

Figure 2

Segregation of the ALS gene harboring W548L/S627I mutations and hyPBase expression vector in T₁ progeny. (a–c) Southern blot analysis with probe 1 (a), 2 (c), 3 (b) shown in Figure 1(a,b) using MfeI-digested genomic DNA of wild-type, a regenerated plant of ALS GT-A and ALS GT-A_hy-10, and four T₁ progeny of ALS GT-A_hy-10 (ALS GT-A_hy-10-1, 2, 3, and 4). Southern blot analysis revealed that the ALS locus with W548L/S627I segregated in T₁ progenies of ALS GT-A_hy-10 (wild-type ALS gene, line no. 1; heterozygous modification of the ALS gene, line no. 2 and 3; homozygous modification of ALS gene, line no. 4). (d) Schematic diagram of hyPBase expression vector. The hyPBase expression vector carries a rice Ubi-1 promoter (Pubi)::hyPBase (PBase) cassette and a rice actin promoter (Pact)::nptII expression cassette. T, terminator; LB, left border; RB, right border. (e) Southern blot analysis with probe 4 shown in (d) using SpeI-digested genomic DNA of wild-type, a regenerated plant of ALS GT-A and ALS GT-A_hy-10, and four T₁ progeny of ALS GT-A_hy-10 (ALS GT-A_hy-10-1, 2, 3, and 4). The hyPBase expression vector segregated out in ALS GT-A_hy 10-3.

Figure 3

Analysis of the ALS gene harboring W548L/S627I mutations in T₁ progeny. (a) Diagram showing the targeted ALS locus. Arrowheads indicate the primers used for the reverse transcriptase-polymerase chain reaction (RT-PCR, b) and CAPS (c). The expected band sizes of the RT-PCR (0.9-kb) and CAPS (wild-type, 0.9-kb; GT 0.7-kb and 0.2-kb) are shown. (b) Transcript levels of the ALS gene in T₁ plants carrying the wild-type (line no. 1) and modified ALS gene (heterozygous, lines no. 2, 3 or homozygous, line no. 4). Top and middle panels show RT-PCR analysis using the ALS gene-specific primers with (top) or without (middle) reverse transcriptase (RT-Ace + or −, respectively). The Actin 1 (Act1) gene was used as an internal control (bottom panel). (c) CAPS analysis combining PCR analysis using ALS gene-specific primers with cDNA (top) or genomic DNA (bottom) and MfeI digestion in T₁ plants carrying the wild-type (line no. 1) or modified ALS gene (heterozygous, lines no. 2, 3 or homozygous, line no. 4). (d) Herbicide bispyribac (BS)-tolerant phenotype of T₁ plants. GT line A_hy T₁ plants carrying the modified ALS gene [either heterozygous (GT-hetero) or homozygous (GT-homo)] showed BS tolerance after 3 weeks of BS treatment, but not T₁ plants carrying the wild-type ALS gene (ALS-wt).