Edit-R HDR Plasmid Donor Kits

Kit for assembly of a plasmid DNA EGFP, mKate2, or custom sequence HDR knock-in donor


Edit-R HDR Plasmid Donor Kits
Kits contain the static components needed to construct a custom plasmid DNA donor to introduce the EGFP or mKate2 fluorescence reporter, or a custom insert sequence to any genomic location using CRISPR-Cas9 and the HDR pathway

Gene engineering using CRISPR-Cas9 relies on two pathways for repair of the cut target DNA, non-homologous end joining (NHEJ) and homology-directed repair (HDR). Precise repair of the DNA to generate a specific mutation or insert or remove a desired sequence requires the HDR pathway. This process is dependent on the availability of a DNA donor template that can be used to repair the double-strand break in the DNA. One of the most common applications is insertion of a fluorescent reporter sequence on one end of a gene. The Edit-R HDR Plasmid Donor Kit enables rapid and easy creation of a plasmid donor for insertion of the EGPF or mKate2 fluorescence reporter or a user-provided custom sequence into almost any genomic location.

Highlights

  • Fast and efficient assembly of a DNA donor plasmid
  • Validated protocols for design, assembly, and confirmation of the donor plasmid
  • Utilize the CRISPR Configurator for designing the crRNA to the cut site 
  • Seamless integration with the HDR Donor Designer for designing the primers to create the required 5′ and 3′ homology arms flanking the insertion site

The HDR plasmid donor kit contains all the DNA components to build and quality check a custom DNA donor template plasmid to introduce a fluorescent reporter to almost any genomic location when coupled with Edit-R HDR DNA donor plasmid homology arm custom primer pairs.

The HDR Donor Designer is used to design and order the PCR primers for the amplification of the 5′ and 3′ homology arms, required for assembly of the plasmid.

Kit Components & Amounts

Cat # Description Item # Amount
UK-008100-01-10 Edit-R HDR Plasmid Donor Kit - EGFP    
  Edit-R HDR Plasmid Donor Backbone U-008300-01-1200 1200 ng
  Edit-R HDR insert - EGFP U-008100-01-250 250 ng
  Edit-R Colony PCR Primer EGFP Forward U-008100-FB-05 5 nmol
  Edit-R Colony PCR Primer EGFP Reverse U-008100-RA-05 5 nmol
  Edit-R Colony PCR Primer Backbone Forward U-008300-FW-05 5 nmol
  Edit-R Colony PCR Primer Backbone Reverse U-008300-RV-05 5 nmol
UK-008200-03-10 Edit-R HDR Plasmid Donor Kit -mKate2    
  Edit-R HDR Plasmid Donor Backbone U-008300-01-1200 1200 ng
  Edit-R HDR insert - mKate2 U-008200-01-250 250 ng
  Edit-R Colony PCR Primer mKate2 Forward B U-008200-FB-05 5 nmol
  Edit-R Colony PCR Primer mKate2 Reverse A U-008200-RA-05 5 nmol
  Edit-R Colony PCR Primer Backbone Forward U-008300-FW-05 5 nmol
  Edit-R Colony PCR Primer Backbone Reverse U-008300-RV-05 5 nmol
UK-008300-01-10 Edit-R HDR Plasmid Donor Kit - Universal    
  Edit-R HDR Plasmid Donor Backbone U-008300-01-1200 1200 ng
  Edit-R Colony PCR Primer Backbone Forward U-008300-FW-05 5 nmol
  Edit-R Colony PCR Primer Backbone Reverse U-008300-RV-05 5 nmol

 

  
HazardousNo
Shipping ConditionAmbient
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-20 C,-80 C
Edit-R™ mKate2 Insert Sequence 
 
LOCUS: Edit-R mKate2 Sequence         
714 bp DNA linear   
            
FEATURES                    Location/Qualifiers
N-terminal flexible linker    1..12
mKate2                            13..705
C-terminal flexible linker    706..714
 
ORIGIN      
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   
 
FEATURES                   Location/Qualifiers
N-terminal flexible linker   1..12
EGFP                              13..726
C-terminal flexible linker   727..735
 
ORIGIN      
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

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

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

mKate2-SEC61B fusion in U2OS cells

mKate2-SEC61B-fusion

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.


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

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

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.


Diagram-of-DSB-repair

Diagram of double-strand break repair

Diagram-of-DSB-repair

Diagram of double-strand break (DSB) repair using the homology-directed repair (HDR) pathway.


Edit-R-mKate2-HDR-donor-map

Dharmacon™ Edit-R™ mKate2 knock-in donor

Edit-R-mKate2-HDR-donor-map

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

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

Detection of successful cloning by colony PCR

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-mKate2-knock-in-cell-line

Workflow to acquire mKate2 knock-in clonal cell line

Workflow-mKate2-knock-in-cell-line

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


Sequence verification of positive clones

Sequence verification of positive clones

Sequence verification of positive clones

Following colony PCR, Sanger sequencing was utilized to confirm the sequences of positive clones.