Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants

Abstract

Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 5'-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants.

Figure 1.

Diagram of the Arabidopsis BFP-to-GFP transgenic system for detecting ssODN-mediated precision genome editing. The edited nucleotide change (C→T) that results in the conversion from BFP to GFP fluorescence is shown as blue and green letters, respectively.

Figure 2.

Effects of ssODNs on BFP-to-GFP editing in Arabidopsis protoplasts pretreated with phleomycin. Protoplasts were pretreated with 0, 250, or 1,000 µg mL⁻¹ phleomycin for 90 min prior to the delivery of ssODN BFP/41 or BFP/41/NT. GFP fluorescence was measured by cytometry 24 h after the delivery of ssODNs. Data represent means ± se (n = 4).

Figure 3.

BT-1 TALEN design and target diagram. A, The mannopine synthase (MAS) promoter drives the expression of the TALEN monomers. The pea (Pisum sativum) rbcT RBCSE9 acts as a gene terminator. A V5 epitope tag and an SV40 nuclear localization signal (NLS) reside on the N terminus. B, BFP target region schematic. The BT-1 TALE left and right binding domains are underlined. The site of BFP-to-GFP conversion (C→T) is in blue. BT-1 activity produces a DSB that can be repaired by NHEJ or through ssODNs, resulting in indels or BFP-to-GFP precision editing, respectively. Red bases are silent substitutions used to discourage BT-1 activity after conversion.

Figure 4.

Addition of ssODNs enhances genome editing in Arabidopsis protoplasts treated with TALEN BT-1. A, Imprecise NHEJ repair events in Arabidopsis protoplasts treated with BT-1 TALEN. B, Percentage of total indels as a result of BT-1 activity by length. C, Addition of ssODN to BT-1 TALEN-treated protoplasts significantly enhances the frequency of BFP-to-GFP conversion in Arabidopsis. Arabidopsis protoplasts were treated with BFP/41, BFP/101, or BFP/201 with and without BT-1 TALEN or BT-1 TALEN without ssODN. BFP-to-GFP edits were evaluated by cytometry 72 h after delivery. Data represent means ± se (n = 3).

Figure 5.

CRISPR/Cas9 construct design and target region diagram. A, The MAS promoter drives the transcription of the plant codon-optimized SpCas9 gene that contains two SV40 NLSs at the N and C termini and a 3× FLAG epitope tag on the N terminus. The pea rbcT RBCSE9 acts as a gene terminator. The AtU6-26 promoter drives the transcription of the poly(T) terminated sgRNA scaffold. B, Approach used to target the BFP transgene using BC-1 CRISPR/Cas9. The protospacer is shown as a black line and the protospacer adjacent motif (PAM) as a red line. The edited nucleotide change (C→T) resulting in the conversion from BFP to GFP is shown as blue and green letters, respectively. Red nucleotides are silent mutations used to deter BC-1 activity on a converted GFP transgene.

Figure 6.

BC-1 CRISPR/Cas9 activity in Arabidopsis protoplasts. A, Imprecise NHEJ repair events in protoplasts treated with BC-1 as determined by amplicon deep sequencing (n = 1). B, Activity of TALEN BT-1 contrasted with CRISPR/Cas9 BC-1 in Arabidopsis protoplasts as determined by the percentage of imprecise NHEJ events. C, Off-target analysis for BC-1 CRISPR/Cas9. Imprecise NHEJ events at five loci homologous to the BC-1 target sequence were measured by amplicon deep sequencing (n = 1). Bases in lowercase red letters are mismatches to the BC-1 target sequence. D, ssODNs enhance BFP-to-GFP editing in Arabidopsis protoplasts treated with BC-1. Protoplasts were treated with BFP/41 or BFP/101 with and without CRISPR/Cas9 or BC-1 CRISPR/Cas9 alone. BFP-to-GFP edits were measured by flow cytometry 72 h after delivery. Data represent means ± se (n = 5).

Figure 7.

Approach used to target the EPSPS loci in flax. A, Target region of the EPSPS loci Thr-178 and Pro-182 of exon 2. The EC-2 protospacer is shown as a black line, and the PAM is shown as a red line. The nucleotides within the codons targeted for edit are in blue (ACA and CCG), and edited nucleotides are in red (ATA and GCG). The CCG→GCG edit disrupts the PAM, minimizing EC-2 activity on an edited gene. B, Stages in the flax genome-editing workflow. Image 1, Protoplasts (bar = 10 µm); image 2, microcolony at 3 weeks (bar = 50 µm); image 3, microcalli at 7 weeks (bar = 100 µm); image 4, shoot initiation from callus (bar = 0.5 cm); image 5, regenerated shoots (bar = 0.5 cm); and image 6, regenerated plant in soil.

Figure 8.

Sequence confirmation of the edited EPSPS alleles in regenerated plants A23 and B15. The arrow shows a gene-specific single-nucleotide polymorphism (SNP). The boxed areas show the T178I and P182A edits. Regenerated plant A23 contains the T178I and P182A precise edits in one allele of EPSPS gene 2. Regenerated plant B15 contains the T178I and P182A precise edits in one allele of EPSPS gene 1. Chromatograms are representative of multiple genomic DNA extractions from each plant. wt, Wild type.

Figure 9.

Targeted EPSPS edits provide herbicide tolerance to flax calli and regenerated plants. A, Flax wild-type (Wt) calli and calli derived from event A23, which contains T178I and P182A edits in EPSPS gene 2, were cultured in six-well dishes on medium containing a range of glyphosate concentrations. Images were captured 14 d after the initiation of treatment. Each image corresponds to one well. B, Mean fresh weight per well of wild-type and A23 calli treated with glyphosate after 21 d. Data represent means ± se (n = 3). C, Greenhouse-hardened wild-type and A23 whole plants in soil were treated with 10.5 or 21 mm glyphosate or surfactant only by spray application. Images were captured 6 d after glyphosate application. This experiment was repeated multiple times with similar results. Bar = 2 cm.