U2OS cells were transfected with DharmaFECT Duo, Cas9 mRNA, synthetic crRNA-tracrRNA targeting LMNA, synthetic crRNA-tracrRNA targeting SEC61B, an EGFP donor plasmid specific to the N-terminus of LMNA containing ~1000 bps of homology per homology arm, and a mKate2 donor plasmid specific to the N-terminus of SEC61B containing ~1000 bps of homology per homology arm. Cells were passaged as needed, and imaged 7-days post transfection on an InCell 2200 high content fluorescent microscope to identify single cells that contained both EGFP and mKate2 insertion.
Gibson cloning results of the Edit-R donor plasmid kit with universal backbone, EGFP insert, and two PCR generated homology arms. The resulting cloning reaction was transformed in NEB 5-alpha chemically competent cells and grown at 37 degrees C overnight. 10 bacterial colonies were selected at random and colony PCR was performed to detect the presence of each homology arm (L=5’ homology arm and R=3’ homology arm). Colony PCR amplicons were run on a 2% agarose E-gel containing ethidium Bromide and a FastRuler low molecular weight molecular weight marker.
The Edit-R HDR plasmid donor is created through plasmid cloning and assembly of a fluorescent protein insert sequence, the cut vector backbone, and two homology arms generated with custom PCR primers to geneomc regions flanking the insertion site.
Diagram of the plasmid donor cloning workflow including component ordering, homology arm amplification, and plasmid donor generation. Colors on the diagram indicate the origin of the DNA (dark blue = 5' homology arm, light blue = 3' homology arm, red = mKate2 fluorescent reporter sequence, orange = plasmid backbone). medium alone = media-only negative PCR control.
Workflow showing the major process steps and general timing necessary to generate a mKate2 knock-in clonal cell line.
Edit-R mKate2 plasmid donor map with important vector features. The example includes 500 bp homology arms for reference.
Following colony PCR, Sanger sequencing was utilized to confirm the sequences of positive clones.