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

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

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

Fig. 2

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.

Back to article page