LMNA tagged at the N-terminus with EGFP in U2OS cells

U2OS cells were transfected with DharmaFECT Duo, Cas9 mRNA, synthetic crRNA-tracrRNA targeting LMNA, and an EGFP donor plasmid specific to the N-terminus of LMNA 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.

mKate2-SEC61B fusion in U2OS cells

Creation of an mKate2 N-terminal fusion to the SEC61B gene, a component of protein transport in the endoplasmic reticulum. U2OS cells were transfected with 50 nM SEC61B-crRNA:tracrRNA, 25 nM Cas9 protein, 2 ng/μL SEC61B mKate2 HDR donor plasmid, and 0.3 μL/well DharmaFECT Duo and visualized on a fluorescent microscope seven days post-transfection. Note: Ribonucleoprotein(RNP) complex ratio is 2:1 for Cas9 protein transfection.

Materials required for cloning HDR plasmid donors

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.

Dharmacon Edit-R mKate2 knock-in donor

Edit-R mKate2 plasmid donor map with important vector features. The example includes 500 bp homology arms for reference.

Diagram of the plasmid donor cloning workflow

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 to acquire mKate2 knock-in clonal cell line

Workflow showing the major process steps and general timing necessary to generate a mKate2 knock-in clonal cell line.