Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

Abstract

Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homology-directed repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and the Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric protein combines the functions of Cas9, which produces targeted and specific DNA double-strand breaks (DSBs), and the VirD2 relaxase, which brings the repair template in close proximity to the DSBs, to facilitate HDR. We successfully employed our Cas9-VirD2 system for precise ACETOLACTATE SYNTHASE (OsALS) allele modification to generate herbicide-resistant rice (Oryza sativa) plants, CAROTENOID CLEAVAGE DIOXYGENASE-7 (OsCCD7) to engineer plant architecture, and generate in-frame fusions with the HA epitope at HISTONE DEACETYLASE (OsHDT) locus. The Cas9-VirD2 system expands our ability to improve agriculturally important traits in crops and opens new possibilities for precision genome engineering across diverse eukaryotic species.

Fig. 1. Chimeric fusion of Cas9 and VirD2 can facilitate precise genome engineering.

Experimental scheme showing that the Cas9 endonuclease and VirD2 DNA binding activities can be exploited for precise genome engineering of plants. The VirD2 protein in the chimeric Cas9-VirD2 fusion will process and covalently bind the RB-edged ssDNA repair templates. The availability of the repair templates in the proximity of the DNA DSBs created by the sgRNA-guided Cas9 endonuclease may enhance the rate of precise repair via the targeted HDR pathway. Alternatively, the NHEJ pathway will imprecisely repair the DNA DSBs.

Fig. 2. Chimeric Cas9-VirD2 fusions efficiently bind to repair template DNA and cleave the DNA target.

a Expression and purification of Cas9-VirD2 and VirD2-Cas9 from BL21(DE3) cells. The HIS column-purified Cas9-VirD2 and VirD2-Cas9 fusion proteins were separated on SDS-PAGE. Both Cas9-VirD2 and VirD2-Cas9 with the exact size of 216 kDa were purified and 1 µg was loaded into the gel for separation. b Confirmation of the nicking and relaxase activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T-DNA vector with the RB and LB was incubated with increasing concentrations of Cas9-VirD2 and VirD2-Cas9. The complex was separated on a 1% TBE agarose gel. The red arrowhead indicates the conversion of the supercoiled plasmid to a completely relaxed gel-retarded DNA structure. c Confirmation of the covalent binding of Cas9-VirD2 and VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) RB sequence (T-RB-60b) and without RB (T-NRB-60b) were incubated with Cas9-VirD2 and VirD2-Cas9 in Mg²⁺ buffer. After incubation, the sample mixture was boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB-60b) to Cas9-VirD2 and VirD2-Cas9. (d) Optimization of the covalent binding of VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) with RB sequence (T-RB) and without RB sequence (T-NRB) were incubated with VirD2-Cas9 in Mg²⁺ buffer. After incubation for 5 min Exonuclease 1 was added to the sample mixture and incubated for another 25 min, the samples were boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB) to VirD2-Cas9. (e and f) Confirmation of the targeted endonuclease activity of Cas9-VirD2 and VirD2-Cas9. The purified Cas9-VirD2 or VirD2-Cas9 proteins and sgRNA with and without repair template were incubated with the target DNA. The reaction mixture was separated on a 2% agarose gel. Arrowheads indicate the proper digestion of the target by the Cas9-VirD2 and VirD2-Cas9-sgRNA complex in the presence and absence of the repair templates.

Fig. 3. Chimeric Cas9-VirD2 fusions mediate efficient editing of ALS in rice calli.

a Co-immunoprecipitation of the repair template with VirD2-Cas9 from rice protoplasts. Rice protoplasts transfected with Cy5-labeled repair templates (T-RB, templates with RB and T-NRB, templates without RB) and plasmid expressing VirD2-Cas9 using PEG-mediated delivery. VirD2-Cas9 was immunoprecipitated with anti-Flag bound agarose beads. Part of each sample was run on a 6% agarose gel and imaged (upper panel) for Cy5. The unlabeled (T-RB) and Cy5-labeled (Cy5-T-RB) repair templates were used as controls. A part of the sample was run on SDS-PAGE and the immunoblot was developed for the Flag tag (VirD2-Cas9-Flag) as input with anti-Flag and anti-mouse secondary antibody. b Schematic diagram of the in planta expression plasmids. The plant codon-optimized Cas9-VirD2 and VirD2-Cas9 complexes were cloned into a plant expression vector under the control of the Ubiquitin promoter followed by a terminator, and sgRNAs were cloned under the U6 promoter followed by a termination signal. c Confirmation of the in vivo endonuclease activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T7EI assay was conducted with 200 ng of target-flanking purified PCR product. A high rate of Indels was detected by the T7EI digestion assay of the samples expressing Cas9 (58%), Cas9-VirD2 (53%), or VirD2-Cas9 (47%) and sgRNA. T7EI-treated samples were separated on a 2% agarose gel. Arrowheads indicate the corresponding T7EI digestion at the Indel created by the endonuclease activity of the Cas9 fusions. d Alignments of the Sanger sequencing reads of the target-flanked PCR product cloned into the pJet2.1 plasmid. The top line shows the wild-type sequence with the target sequence in green and the PAM sequence underlined. Alignment analysis shows the presence of Indels at the target sites. The induced Indels are represented by numbers to the right of each lane.

Fig. 4. Chimeric Cas9-VirD2 fusions with phosphorothioate-modified repair templates make efficient edits at the ALS target locus.

a Schematic of the desired edit (green) in the wild-type OsALS allele. The desired modification (W548L), the MfeI recognition site, and the modified PAM sequence in the repair template are shown. The repair template (red line, right and left homology arms) with the desired modification (green) was used to make edits at the target site (black line). The forward allele-specific PCR primer is represented by an arrow corresponding to the desired modification and the reverse primer outside the homology arm sequence. b Allele-specific confirmation of successful editing in the rice callus. The allele-specific PCR from the DNA extracted from 24 individual calli bombarded with phosphorothioate-modified repair templates and unmodified repair templates with and without RB sequence (T-NRB, mT-NRB, T-RB and mT-RB) with Cas9 or Cas9-VirD. The phosphorothioate-modified repair templates are more stable than the unmodified repair templates and enhance the rate of HDR in combination with Cas9-VirD2. The amplification of the exact size 417 bp fragment only in the VirD2-Cas9 or Cas9-VirD2 with mT-RB samples by allele specific PCR (indicated by the arrow head) confirmed the presence of the herbicide resistance allele. The lower band is a nonspecific PCR amplification and present in all samples. c Molecular confirmation of the exact insertion of the desired nucleotide sequence at the target locus via MfeI digestion. Restriction digestion of the target-flanked PCR product from the DNA of pooled selected calli (N = 100). Using ImageJ software, the rate (in %) of the exact repair (MfeI recognition site insertion) was calculated and is represented below the corresponding lane. Cas9 alone was used as the baseline control. The arrowheads represent the MfeI-digested fragments.

Fig. 5. The Cas9-VirD2-coupled HDR system efficiently inserted the HA epitope into the C terminus of OsHDT.

a Schematic of the in-frame insertion of the HA epitope at the C terminus of OsHDT. The short right and left homology arms (blue) flanked the HA epitope (green). The DNA DSB is shown by a line, and the site of the insertion is indicated by a black arrow. The dotted lines represent the targeted HDR site. The forward allele-specific PCR primer corresponding to the HA epitope DNA sequence and the reverse primer outside the homology arm sequence are represented by red arrows. b Confirmation of the insertion of the HA epitope at the C-terminal end of OsHDT. The allele-specific PCR (22 cycles) from the DNA extracted from pooled (N = 100) calli bombarded with the end modified repair template (left homology arm–HA epitope–right homology arm) with and without the RB (mT-RB and mT-NRB) and Cas9-VirD2, VirD2-Cas9, or Cas9. c Representation of the enhanced rate of insertion of the HA epitope using the repair templates containing the RB sequence and Cas9-VirD2 or VirD2-Cas9. d Confirmation of the targeted insertion of the HA epitope at the C terminus of OsHDT via Sanger sequencing. The HA epitope-specific forward primer PCR product from the DNA of the pooled calli (N = 24) was cloned into the pJet2.1 plasmid and subjected to Sanger sequencing. The exact alignment of the Sanger sequence reads confirmed the successful editing at the C-terminal end of OsHDT in the calli bombarded with the repair templates with the RB and VirD2-Cas9. Box in Sanger sequencing chromatogram represent the precise repair and accurate end junction of the repair template insertion at c-terminus of OsHDT.

Fig. 6. Engineering herbicide resistance in rice.

a Confirmation of the presence of the herbicide resistance allele in regenerated rice plants. Genomic DNA was extracted from individual regenerated plants, and the target-flanked PCR-amplified fragment was subjected to MfeI digestion. MfeI digestion confirmed a high rate of successful editing in the plants regenerated from calli bombarded with repair templates containing the RB sequence and Cas9-VirD2 or VirD2-Cas9. b, c Alignment of the Sanger sequencing reads and representative chromatograms of the plants that had been confirmed by MfeI digestion. The PCR-amplified fragments from the individual plants were cloned into the pJet2.1 plasmid and subjected to Sanger sequencing. The repair-specific nucleotide modifications are shown in blue, and their exact locations are indicated by arrowheads (red). A representative chromatogram also shows the exact repair at the targeted locus.

Fig. 7. Heritability of the engineered herbicide resistance in rice plants.

a Confirmation of the herbicide resistance in the T₁ progenies of rice plants. Seeds collected from line 70 and line 121 were germinated on vertical plates with 1/2 MS media containing bispyribac. Seeds from the plants expressing only Cas9 were used as a control. Plants with the modified ALS allele showed proper root and shoot development. Plants expressing only Cas9 with repair templates (CNT) were used as control. b Molecular confirmation of the modified ALS allele. Genomic DNA was extracted from individual T₁ plants, and the target-flanked PCR-amplified fragment was subjected to MfeI digestion. MfeI digestion confirmed the proper conversion of the ALS allele to the herbicide-resistant allele in the selected plants. Arrowheads indicate the MfeI-digested fragment. c Alignment of the Sanger sequencing reads of the selected lines 70-1, 70-2, 121-02, and 121-11. The PCR-amplified fragments from the individual plants were cloned into the pJet2.1 plasmid and subjected to Sanger sequencing. The exact locations of the specific nucleotide modifications are indicated by red arrowheads.

Fig. 8. Efficient engineering of plant architecture through HDR.

a Schematic of the desired mutations in the wild-type OsCCD7 allele. Modifications of nucleotides added into the repair templates at the sgRNA binding and PAM sequences to avoid retargeting of the repaired sequence by VirD2-Cas9 are shown. The sgRNA binding sequence is indicated by an arrow and removal of the PAM and insertion of the EcoRI recognition sequence (underlined) is indicated by a line. Nucleotide modifications along with the corresponding amino acids are represented by capital, colored, and bold letters. The respective wild-type amino acids (capital and bold) are also shown. b Representative gels of EcoRI digestion for HDR confirmation. Target (CCD7) flanking 445 bp PCR amplified fragments were subjected to EcoRI digestion. EcoRI digestion confirmed the proper digestion to produce 270 + 175 bp fragments (indicated by arrowheads) and repair in the plants regenerated from calli bombarded with repair templates with mT-RB and Cas9-VirD2 or VirD2-Cas9. Repair templates without mT-NRB with VirD2-Cas9 and Cas9 with mT-RB repair templates were used as control. Lines confirmed with EcoRI digestion are indicated with asterisks. c Photos of plants with engineered modifications. Seed of the progeny plants were grown on soil in the greenhouse and pictures were taken 40 days after germination. d Heritability of the repaired modifications. The target (CCD7) flanking PCR amplified 445-bp fragments from the progeny plants of the selected lines were subjected to EcoRI digestion. EcoRI digestion produced 270 + 170 bp fragments in lines that had inherited the modification. Four out seventeen plants are homozygous for the EcoRI site. e Alignment of the Sanger sequencing reads and representative chromatograms of the EcoRI digestion of confirmed homozygous plants. The repair-specific nucleotide modifications are shown in blue and their exact locations are indicated by arrowheads (red). The representative chromatograms show the exact repair at the targeted locus.