Edit-R™ mKate2 Insert Sequence
LOCUS: Edit-R mKate2 Sequence
714 bp DNA linear
N-terminal flexible linker 1..12
C-terminal flexible linker 706..714
1 gctggtggca gcgtgagcga gctgattaag gagaacatgc acatgaagct gtacatggag
61 ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
121 ggcacccaga ccatgagaat caaggcggtc gagggcggcc ctctcccctt cgccttcgac
181 atcctggcta ccagcttcat gtacggcagc aaaaccttca tcaaccacac ccagggcatc
241 cccgacttct ttaagcagtc cttccccgag ggcttcacat gggagagagt caccacatac
301 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
361 tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa
421 acactcggct gggaggcctc caccgagacc ctgtaccccg ctgacggcgg cctggaaggc
481 agagccgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa cttgaagacc
541 acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac
601 agaagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca gcacgaggtg
661 gctgtggcca gatactgcga cctccctagc aaactggggc acagaggagg tagc
Edit-R™ EGFP Insert Sequence
LOCUS Edit-R EGFP Sequence
735 bp DNA linear
N-terminal flexible linker 1..12
C-terminal flexible linker 727..735
1 gctggtggca gcgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
61 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
121 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
181 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
241 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
301 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
361 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
421 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
481 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
541 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
601 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
661 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
721 tacaagggag gtagc
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.
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.
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.