High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

Abstract

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

Keywords: CRISPR/Cas9; DNA replicons; Wheat; genome editing; homologous recombination; multiplexed gene targeting; technical advance.

Figure 1.

Replication cycle of wheat dwarf virus (WDV)-derived replicons in transformed plant cells. For genome engineering purposes, the WDV-derived replicon is delivered into plant cell nuclei by either particle bombardment (double-stranded DNA, dsDNA) or Agrobacterium-mediated transformation (single-stranded DNA, ssDNA). Then, an ssDNA replicon is released and converted into dsDNA by host polymerases. The replication initiation protein (Rep) recognizes a domain in the large intergenic region (LIR) and nicks the DNA at a 9-nt conserved site found on the hairpin structure of the LIR to promote rolling circle replication (RCR). As a result, newly synthesized ssDNA replicons are formed and converted again into dsDNA replicons. The new dsDNA replicons can be used to either express the encoded proteins (Rep, RepA and heterologous proteins such as GFP) or start a new RCR cycle.

Figure 2.

Wheat dwarf virus (WDV) replicon replication and performance of different WDV architectures designed for genome engineering of cereal species. (a) Time course of WDV replication and relative transcript level of GFP and Rep/RepA proteins in wheat calli at different days post bombardment (dpb). (b) Normalized copy number of the GFP-containing replicon relative to the Ubi-GFP control at 5 dpb. (c) GFP expression in wheat calli using the different WDV architectures: WDV1-GFP (left panel), WDV2-GFP (central panel) and WDV3-GFP (right panel). (d) qRT-PCR quantification of transcript level normalized to the non-replicating pWDV3-GFP. (e) Replicon copy number normalized to the non-replicating pWDV3-GFP and detection of circularization of the replicon and the actin PCR control. The colored arrows in (c) indicate the primers used to detect circularization in each replicon variant. Error bars represent the standard errors (SEs) of three independent biological replicates (n = 6 wheat calli per replicate) transformed by particle bombardment. Gold particles with no DNA were used to transform the wild-type (WT) control.

Figure 3.

Wheat dwarf virus (WDV) replicon-mediated expression of the CRISPR/Cas9 system for targeted mutagenesis in wheat cells. (a) CRISPR/ Cas reagents expressed from the different architectures of the WDV replicons. (b) PCR/restriction enzyme assay to detect mutations in the ubiquitin gene induced with the sgUbi1 expressed in the different vectors shown in (A); *percentages of NHEJ (% ±SEs) represent the average of two different transfections and have been normalized to the transfection efficiency (40 and 60%, respectively). (c) Sequences of resistant bands obtained by NHEJ of the ubiquitin gene mediated by the pWDV1-CR vector.

Figure 4.

High-efficiency gene targeting (GT) mediated by wheat dwarf virus (WDV)-derived replicons expressing CRISPR/Cas9 reagents in wheat cells. (a) GT-mediated expression of GFP in wheat protoplasts 2 days after transfection with the WDV vectors carrying the CRISPR/Cas9 reagents and the T2A:gfp donor template. Quantification of the number of GFP-positive cells was carried out by flow cytometry. (b) Schematic of the expected integration of the T2A:gfp sequence in the genomic ubiquitin locus and molecular characterization by PCR using specific primers. Lane numbers in the gel denote the following constructs: 1, pWDV1.CR.GFP; 2, pWDV2.CR.GFP; 3, pWDV3.CR.GFP; (c), DNA sequences for the 5' and 3' junctions of the integrated T2A:gfp fragment obtained with the pWDV1.CR.GFP vector. (d) Enhancement of GT efficiency in wheat scutella mediated by the WDV-derived replicons. Black bars denote the normalized GT efficiency compared with the non-viral control (pCR.GFP, blue bar). Error bars represent the standard error (SE) of five different transformation experiments (n = 132 scutella for each treatment). Analysis of variance (ANOVA) was performed. Different letters in the bar graph indicate significant differences (P < 0.05), as determined by the ‘least significant difference’ (LSD) post-hoc all-pairwise comparison test. Replicon circularization and endogenous actin gene control were detected by PCR. Total GT frequency (% ±SE) represents the percentage of cells with GT events relative to the total number of transformed cells (as calculated with the pWDV1-GFP control in each individual experiment). The average percentage of scutella showing at least one GT event is shown in parenthesis.

Figure 5.

Single-cell multiplexed gene targeting (GT) mediated by the wheat dwarf virus (WDV)-CRISPR/Cas9 system in wheat cells. (a) GT-mediated expression of BFP in wheat protoplasts 2 days after transfection with the WDV vectors carrying the CRISPR/Cas9 reagents and the P2A:bfp donor template. Quantification of the BFP-positive cells was carried out by image quantification. (b) Schematic of the expected integration of the P2A:bfp sequence in the genomic MLO locus and PCR amplification of the 5' and 3' junctions using specific primers. (c) DNA sequences are shown for the 5' and 3' junctions, indicating the knock-in of the P2A:bfp into the MLO homoeoallele in the D genome. (d) Multiplexed GT of the promoter-less T2A:gfp and P2A:bfp sequences in wheat protoplasts. GFP and BFP channels are shown in the left-hand and center pictures, respectively. The right-hand picture is a merged image of the two single-channel pictures. The arrow in the right picture denotes a single cell expressing both GFP and BFP. (e) Diagram of GFP, BFP and GFP+BFP knock-in frequencies relative to the total number of cells that have undergone GT.