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.

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.

U2OS cell line with EGFP-LMNA and mKate2-SEC61B fluorescent reporters generated through multiplexed CRISPR-Cas9-mediated knock-in

Briefly, synthetic crRNAs targeting LMNA and SEC61B were designed to generate double-strand breaks near the N-termini of each of the proteins using the CRISPR RNA Configurator. The Edit-R HDR Donor Designer was used to design reagents to generate EGFP and mKate2 donor plasmids specific to the N-termini of LMNA and mKate2, respectively. Both donor plasmids contained ~1000 base pairs of homology per homology arm. U2OS cells were plated at 10,000 cells per well in a 96-well plate the day before transfection. Cells were transfected using 0.3 µL/well DharmaFECT Duo transfection reagent according to optimized conditions determined in previous experiments. The following reagents were transfected into the cells simultaneously: 200 ng Edit-R™ Cas9 Nuclease mRNA, 25 nM synthetic crRNA:tracrRNA targeting LMNA, 25 nM synthetic crRNA:tracrRNA targeting SEC61B, 200ng EGFP-LMNA donor plasmid and 200 ng mKate2-SEC61B donor plasmid. Cells were passaged three times and imaged 7-days post transfection on a Nikon inverted epifluorescent microscope to identify single cells that contained both EGFP (green) and mKate2 (red) insertion.

LMNA tagged at the N-terminus with EGFP and SEC61B tagged at the N-terminus with mKate2 in U2OS cells

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.

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.

Detection of successful cloning by colony PCR

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.

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.