Figure 2. Epitope tagging of ACTB in U2OS cells. The gRNA and adapter primers for C-terminal tagging of human beta-actin gene were designed by TrueDesign Genome Editor and donor DNA prepared using the TrueTag Donor DNAKit, HA. The donor DNA was prepared by one-step PCR using C-3xHA donor DNA templates containing either puromycin or blasticidin selection marker. The resulting PCR product (500 ng) was co-delivered with TrueCut Cas9 Protein v2/gRNA (RNP) complexes (500 ng Cas9/125 ng sgRNA) into 2x105 U2OS cells in a 24-well plate using Lipofectamine CRISPRMAX Cas9 Transfection Reagent. At 48 hours post transfection, the cells were split into 1:4 and then treated with 0, 0.75 µg/ml or 1 µg/ml puromycin or 0, 8 µg/ml, or 12 µg/ml blasticidin for 5 to 7 days. (A) Upon antibiotic selection, aliquots of cells were seeded on cover slips, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with HA Tag Monoclonal Antibody (2-2.2.14), DyLight 488. The Texas Red-X Phalloidin and NucBlue Live ReadyProbes Reagent were used to label the cytoskeleton and nucleus of the cells. The cells were then imaged using Thermo Scientific CellInsight CX7 High Content Analysis (HCA) Platform. (B) After expansion in 6-well plates, the cells were harvested and lysed in approximately 100 µl of Pierce IP Lysis Buffer supplemented with protease inhibitor cocktail. Aliquot of 20 µl supernatant was analyzed by NuPAGE 4–12% Bis-Tris Protein Gels. Untransfected cell lysate was used as negative control. The fractionated proteins were transferred to a nitrocellulose membrane using iBlot 2 Gel Transfer Device. The membrane was blocked for 30 to 60 minutes and then incubated overnight with HA Tag Monoclonal Antibody (2-2.2.14) at 1:1,000 dilution. After washing, the membrane was incubated with the Goat anti-mouse secondary antibody conjugated to HRP at 1:2,000 dilution for 30–60 minutes. Upon extensive washing, the membrane was developed with SuperSignal West Dura Extended Duration Substrate and imaged with iBright. Antibodies against GAPDH were used as loading controls.
Easily create transfection-ready donor DNA for gene tagging
Invitrogen design tools and reagents offer a fast and easy way to create donor DNA for fluorescent tags, epitope tags, or precise gene editing experiments using CRISPR-Cas9 or TALEN technology. Invitrogen TrueTag Donor DNA kits maximize editing efficiency and significantly reduce protocol time compared to competitor products because cloning steps have been eliminated. The kit comes complete with all the necessary reagents to prep the donor DNA and easy-to-follow protocols help ensure that even novice users can perform successful knock-ins in your own lab.
- Achieve up to 100% edited cells—get great results, regardless of your level of expertise
- Eliminate cloning steps—one-step PCR gives you donors in hours, not days
- Minimize screening time—enrich edited cells using blasticidin or puromycin
- Tag genes easily—add your choice of a fluorescent or epitope tag to a protein of interest
- Create knockout cell lines—combine functional knockout with fluorescence and selection markers to enrich edited cells
For small insertions/deletions (up to 30 bases), we recommend using the Invitrogen TrueDesign Genome Editor. This free online software allows you to design and order CRISPR gRNA and ssDNA donors for insertions, deletions, and replacements up to 30 bases.
| Type of genomic edit | TrueDesign Genome Editor | TrueTag Donor DNA Kits |
|---|---|---|
| SNP edit or other replacement of ≤30 nt |  | |
| Deletion or insertion of ≤30 nt | | |
| Insertion of STOP codons for functional knockout | | |
| Insertion of STOP codons, fluorescent tag, and selection marker for knockout + enrichment of edited cells | | |
| Fluorescent or epitope gene tag | | |
TrueTag Donor DNA Kit selection guide for TALEN or CRISPR knock-in experiments
| Fluorescent kits | Epitope kits | Stem kits | Knockout enrichment kit | |
|---|---|---|---|---|
| Purpose of Kit | Fluorescent tagging of an expressed protein | Epitope tagging of an expressed protein | Fluorescent tagging of an expressed OR non-expressed gene | Functional knockout of a target gene Knock-in of STOP codons & fluorescent tag |
| Tags available | GFP, RFP, BFP or YFP | Myc, HA, 6xHis, DDK (DYKDDDDK; FLAG® Tag) | GFP or RFP | GFP & RFP |
| Antibiotic resistance for enrichment of edited cells | Puromocyin or blasticidin | |||
| Cre/Lox removal of resistance marker following enrichment | | | | |
| Fusion tag expression | | | | |
| Co-expression of fluorescent tag | | |||
| Antibody detection of tag/edit | | |||
| FACS analysis/enrichment of edited cells | | | | |
| Order | Order | Order | Order | |
Gene tagging sample data using TrueTag DNA Donor Kits
Data: Add a fluorescent tag at the C- or N-terminus of an endogenous protein
The TrueTag Donor DNA kits offer four choices of fluorescent tags (EmGFP, tagRFP, tagBFP, tagYFP) for straightforward creation of reporter cell lines to understand protein localization. Tags may be added to either the N- or the C-terminus of the endogenous gene and include a selection marker for enrichment of edited cells.
Figure 1. TrueTag knock-in to U2OS cells. U2OS cells were transfected with TrueCut Cas9 v2, a TrueTag dsDNA donor for homology directed repair (HDR) to insert GFP at the N-terminal or C-terminal of the ACTB locus, and a TrueGuide gRNA for either the N-terminal or C-terminal of ACTB. Transfection was performed with Lipofectamine CRISPRMAX reagent. Seven days after transfection, the cells were counterstained with NucBlue Live ReadyProbes Reagent and images were captured on an EVOS FL Color Imaging System.
Data: Epitope tagging of ACTB in U2OS cells
Epitope tags are used for detection and purification using a monoclonal antibody specific to the epitope tag. Tagging with an epitope is useful when working with novel proteins or targets for which a reliable antibody is not available. TrueTag Donor DNA kits offer tags expressing human influenza hemagglutinin (HA), c-Myc derived protein (Myc), polyhistidine (6xHis), and the FLAG octopeptide DYKDDDDK (DDK).
Data: Combine RFP and GFP tags to track two proteins in the same cell line
Figure 3. Dual tagging HEK293 cells with GFP and RFP. 293FT cells were transfected with TrueCut Cas9 protein v2, a TrueTag dsDNA donor for homology directed repair (HDR) at the C-terminal of the HIST1H4C locus with GFP-puromycin, a second TrueTag dsDNA donor for HDR at the N-terminal of the ACTB locus with blasticidin-RFP, and TrueGuide Synthetic gRNA targeting each genomic loci. Transfection was performed with Lipofectamine CRISPRMAX reagent. Selective pressure was applied to the 293FT cells to generate a stable pool: five days of blasticidin selection was performed first, followed by five days of puromycin selection. Cells were counterstained NucBlue Live ReadyProbes reagent and images were captures on an EVOS FL Color Imaging System.
Reduce donor DNA preparation from days to hours and eliminate cloning steps
Figure 4. Comparison of the TrueTag Donor DNA Kit workflow and a competitor’s homologous recombination knock in workflow. The TrueTag workflow can be done in a few hours and is as simple as completing a single PCR, column purifying the double-stranded DNA product, and then transfecting the donor (with appropriate CRISPR-Cas9 or TAL effector nucleases) to knock-in the tag into your gene of interest. Conversely, competitor kits require multiple primer designs and PCR steps to assemble the donor molecule into a backbone vector, and subsequent subcloning. This requires additional screening of E. coli colonies as well as sequence verification. The entire process takes several days to complete. The TrueTag system allows for donor molecules to be generated in just a few hours.
C-terminal gene tagging workflow
Figure 5. An overview of the C-terminal gene tagging workflow using TrueTag Donor DNA Kits.
Data: Generate stable CRISPR knock-in cell lines with GFP tags
Creation of stable tagged cell lines is made easier with the selection markers included in all TrueTag Donor DNA templates. Applying selective pressure with blasticidin or puromycin (Figure 6) shows successful generation of a cell population with >99% of cells expressing the GFP tag.
Figure 6. Stable cell lines generated in 10 days. On Day 1, 293FT cells are transfected with TrueCut Cas9 v2, a TrueTag dsDNA donor for homology directed repair at the C-terminal of the ACTB locus to insert GFP-puromycin, and a TrueGuide gRNA for the C-terminal of ACTB, with the Lipofectamine CRISPRMAX reagent. TrueTag donor generation is quick process that takes 3 hours or less to complete. On Day 3 or 4, homology directed repair is complete, and the fusion tag is expressed off the native loci. Approximately 25% of the cells in this example were GFP positive. Selection can begin 72 to 96 hours after transfection. By Day 10, this population of cells stability selected with puromycin is >99% positive for GFP. Data collected on an Attune NxT Flow Cytometer.
Data: Tagged iPSC cells express GFP tag following differentiation into astrocytes
Glial fibrillary acidic protein (GFAP) is a type III intermediate filament protein that is expressed by numerous cell types, including astrocytes during central nervous system development. Using the TrueTag GFP Stem kit, iPS cells were tagged at their GFAP gene, enriched using the selection marker, and differentiated into astrocytes. Upon differentiation, the TrueTag knock-in expressed the GFAP protein GFP tag.
Figure 7. Stable GFP-tagging of GFAP and iPSC differentiation into astrocytes. The endogenous GFAP gene was tagged with EmGFP at its C-terminus in iPSC using the TrueTag Donor DNA Kit, GFP Stem. The resulting GFAP-EmGFP iPSC reporter cell lines were then subjected to PSC neural induction using PSC Neural Induction Medium. The resulting PSCs were then differentiated into astrocyte using astrocyte differentiation medium. (A) A few GFP+ cells were detected at approximately Day 18 of differentiation, whereas most of the cells were GFP-positive at Day 45. (B) To confirm the GFP signal is driven by GFAP gene expression, the cells were stained with anti-GFAP antibody conjugated to eFluor 570. The Red staining confirmed the GFAP expression was overlapped with GFP, indicating that the GFP signal was due to expression of GFAP protein in astrocytes.
Data: Dual-enrichment fluorescent tagging strategy provides 100% knockout population in only ten days
Figure 8. Effective enrichment of cells with HPRT knockout using FACS sorting and dual selection. (Upper) HEK293FT cells were transfected with Cas9 RNP and 250 ng each of GFP-Bsd and RFP-Puro donor DNA targeting human HPRT gene. Donors were prepared using the TrueTag Knockout Enrichment Donor DNA Kit. At 48 hours post transfection, the transfected cells were selected with 0.5 µg/ml Puromycin and 16 µg/ml Blasticidin for ten days to remove the un-integrated cell population. The percentages of GFP+ and RFP+ positive cells were determined by flow cytometric analysis. The GFP/RFP dual positive cells were enriched using a cell sorter. When compared to the parental cell control, the sorted cells exhibited >80% GFP+ and RFP+ cells. (Lower) Western blot results demonstrate HPRT knockdown under varying puromycin/blasticidin treatment and sorted/unsorted cell populations. Under three sets of conditions, when compared to the parental cell control (Neg), the enriched cells achieved 100% knockout of HPRT.
TrueTag Knockout Enrichment Donor DNA Kit workflow
Figure 9. An overview of the knockout enrichment workflow using the TrueTag Knockout Enrichment Donor DNA Kit.
Data: Add a fluorescent tag at the C- or N-terminus of an endogenous protein
The TrueTag Donor DNA kits offer four choices of fluorescent tags (EmGFP, tagRFP, tagBFP, tagYFP) for straightforward creation of reporter cell lines to understand protein localization. Tags may be added to either the N- or the C-terminus of the endogenous gene and include a selection marker for enrichment of edited cells.
Figure 1. TrueTag knock-in to U2OS cells. U2OS cells were transfected with TrueCut Cas9 v2, a TrueTag dsDNA donor for homology directed repair (HDR) to insert GFP at the N-terminal or C-terminal of the ACTB locus, and a TrueGuide gRNA for either the N-terminal or C-terminal of ACTB. Transfection was performed with Lipofectamine CRISPRMAX reagent. Seven days after transfection, the cells were counterstained with NucBlue Live ReadyProbes Reagent and images were captured on an EVOS FL Color Imaging System.
Data: Epitope tagging of ACTB in U2OS cells
Epitope tags are used for detection and purification using a monoclonal antibody specific to the epitope tag. Tagging with an epitope is useful when working with novel proteins or targets for which a reliable antibody is not available. TrueTag Donor DNA kits offer tags expressing human influenza hemagglutinin (HA), c-Myc derived protein (Myc), polyhistidine (6xHis), and the FLAG octopeptide DYKDDDDK (DDK).
Figure 2. Epitope tagging of ACTB in U2OS cells. The gRNA and adapter primers for C-terminal tagging of human beta-actin gene were designed by TrueDesign Genome Editor and donor DNA prepared using the TrueTag Donor DNAKit, HA. The donor DNA was prepared by one-step PCR using C-3xHA donor DNA templates containing either puromycin or blasticidin selection marker. The resulting PCR product (500 ng) was co-delivered with TrueCut Cas9 Protein v2/gRNA (RNP) complexes (500 ng Cas9/125 ng sgRNA) into 2x105 U2OS cells in a 24-well plate using Lipofectamine CRISPRMAX Cas9 Transfection Reagent. At 48 hours post transfection, the cells were split into 1:4 and then treated with 0, 0.75 µg/ml or 1 µg/ml puromycin or 0, 8 µg/ml, or 12 µg/ml blasticidin for 5 to 7 days. (A) Upon antibiotic selection, aliquots of cells were seeded on cover slips, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with HA Tag Monoclonal Antibody (2-2.2.14), DyLight 488. The Texas Red-X Phalloidin and NucBlue Live ReadyProbes Reagent were used to label the cytoskeleton and nucleus of the cells. The cells were then imaged using Thermo Scientific CellInsight CX7 High Content Analysis (HCA) Platform. (B) After expansion in 6-well plates, the cells were harvested and lysed in approximately 100 µl of Pierce IP Lysis Buffer supplemented with protease inhibitor cocktail. Aliquot of 20 µl supernatant was analyzed by NuPAGE 4–12% Bis-Tris Protein Gels. Untransfected cell lysate was used as negative control. The fractionated proteins were transferred to a nitrocellulose membrane using iBlot 2 Gel Transfer Device. The membrane was blocked for 30 to 60 minutes and then incubated overnight with HA Tag Monoclonal Antibody (2-2.2.14) at 1:1,000 dilution. After washing, the membrane was incubated with the Goat anti-mouse secondary antibody conjugated to HRP at 1:2,000 dilution for 30–60 minutes. Upon extensive washing, the membrane was developed with SuperSignal West Dura Extended Duration Substrate and imaged with iBright. Antibodies against GAPDH were used as loading controls.
Data: Combine RFP and GFP tags to track two proteins in the same cell line
Figure 3. Dual tagging HEK293 cells with GFP and RFP. 293FT cells were transfected with TrueCut Cas9 protein v2, a TrueTag dsDNA donor for homology directed repair (HDR) at the C-terminal of the HIST1H4C locus with GFP-puromycin, a second TrueTag dsDNA donor for HDR at the N-terminal of the ACTB locus with blasticidin-RFP, and TrueGuide Synthetic gRNA targeting each genomic loci. Transfection was performed with Lipofectamine CRISPRMAX reagent. Selective pressure was applied to the 293FT cells to generate a stable pool: five days of blasticidin selection was performed first, followed by five days of puromycin selection. Cells were counterstained NucBlue Live ReadyProbes reagent and images were captures on an EVOS FL Color Imaging System.
Reduce donor DNA preparation from days to hours and eliminate cloning steps
Figure 4. Comparison of the TrueTag Donor DNA Kit workflow and a competitor’s homologous recombination knock in workflow. The TrueTag workflow can be done in a few hours and is as simple as completing a single PCR, column purifying the double-stranded DNA product, and then transfecting the donor (with appropriate CRISPR-Cas9 or TAL effector nucleases) to knock-in the tag into your gene of interest. Conversely, competitor kits require multiple primer designs and PCR steps to assemble the donor molecule into a backbone vector, and subsequent subcloning. This requires additional screening of E. coli colonies as well as sequence verification. The entire process takes several days to complete. The TrueTag system allows for donor molecules to be generated in just a few hours.
C-terminal gene tagging workflow
Figure 5. An overview of the C-terminal gene tagging workflow using TrueTag Donor DNA Kits.
Data: Generate stable CRISPR knock-in cell lines with GFP tags
Creation of stable tagged cell lines is made easier with the selection markers included in all TrueTag Donor DNA templates. Applying selective pressure with blasticidin or puromycin (Figure 6) shows successful generation of a cell population with >99% of cells expressing the GFP tag.
Figure 6. Stable cell lines generated in 10 days. On Day 1, 293FT cells are transfected with TrueCut Cas9 v2, a TrueTag dsDNA donor for homology directed repair at the C-terminal of the ACTB locus to insert GFP-puromycin, and a TrueGuide gRNA for the C-terminal of ACTB, with the Lipofectamine CRISPRMAX reagent. TrueTag donor generation is quick process that takes 3 hours or less to complete. On Day 3 or 4, homology directed repair is complete, and the fusion tag is expressed off the native loci. Approximately 25% of the cells in this example were GFP positive. Selection can begin 72 to 96 hours after transfection. By Day 10, this population of cells stability selected with puromycin is >99% positive for GFP. Data collected on an Attune NxT Flow Cytometer.
Data: Tagged iPSC cells express GFP tag following differentiation into astrocytes
Glial fibrillary acidic protein (GFAP) is a type III intermediate filament protein that is expressed by numerous cell types, including astrocytes during central nervous system development. Using the TrueTag GFP Stem kit, iPS cells were tagged at their GFAP gene, enriched using the selection marker, and differentiated into astrocytes. Upon differentiation, the TrueTag knock-in expressed the GFAP protein GFP tag.
Figure 7. Stable GFP-tagging of GFAP and iPSC differentiation into astrocytes. The endogenous GFAP gene was tagged with EmGFP at its C-terminus in iPSC using the TrueTag Donor DNA Kit, GFP Stem. The resulting GFAP-EmGFP iPSC reporter cell lines were then subjected to PSC neural induction using PSC Neural Induction Medium. The resulting PSCs were then differentiated into astrocyte using astrocyte differentiation medium. (A) A few GFP+ cells were detected at approximately Day 18 of differentiation, whereas most of the cells were GFP-positive at Day 45. (B) To confirm the GFP signal is driven by GFAP gene expression, the cells were stained with anti-GFAP antibody conjugated to eFluor 570. The Red staining confirmed the GFAP expression was overlapped with GFP, indicating that the GFP signal was due to expression of GFAP protein in astrocytes.
Data: Dual-enrichment fluorescent tagging strategy provides 100% knockout population in only ten days
Figure 8. Effective enrichment of cells with HPRT knockout using FACS sorting and dual selection. (Upper) HEK293FT cells were transfected with Cas9 RNP and 250 ng each of GFP-Bsd and RFP-Puro donor DNA targeting human HPRT gene. Donors were prepared using the TrueTag Knockout Enrichment Donor DNA Kit. At 48 hours post transfection, the transfected cells were selected with 0.5 µg/ml Puromycin and 16 µg/ml Blasticidin for ten days to remove the un-integrated cell population. The percentages of GFP+ and RFP+ positive cells were determined by flow cytometric analysis. The GFP/RFP dual positive cells were enriched using a cell sorter. When compared to the parental cell control, the sorted cells exhibited >80% GFP+ and RFP+ cells. (Lower) Western blot results demonstrate HPRT knockdown under varying puromycin/blasticidin treatment and sorted/unsorted cell populations. Under three sets of conditions, when compared to the parental cell control (Neg), the enriched cells achieved 100% knockout of HPRT.
TrueTag Knockout Enrichment Donor DNA Kit workflow
Figure 9. An overview of the knockout enrichment workflow using the TrueTag Knockout Enrichment Donor DNA Kit.
TrueTag Donor DNA Kit FAQs
Generating your donor DNA construct requires very little expertise in molecular biology techniques. In addition to the TrueTag kit providing templates, PCR essentials, and clean-up reagents, the TrueDesign Genome Editor software walks you through every design step with a simple and straightforward interface. It will generate the required CRISPR guide RNA, amplification primers with homology arms, and verification primers in just minutes, with convenient Add to Cart functionality.
Yes, the TrueTag system is available with four different fluorescent markers (GFP, RFP, BFP, or YFP) that work with the most common filter sets of microscopes that utilize fluorescent detection. This also allows for simultaneous tagging and visualization of different proteins.
The TrueTag Stem kits are designed for this type of experiment. The selection marker is driven by its own promoter, so enrichment can happen in the absence of the endogenous gene expression, which also drives expression of the fluorescent tag.
You will need to determine this based on the desired outcome of your experiment. If you want to knock out all known alleles, you will need to select a region in a transcript that is shared across all splice variants. If you want to knock out only a certain variant, you will need to choose a region that is unique to that transcript. TrueDesign software links out directly to NCBI for easy access to the most current transcript topology to help you make this decision.
The KO enrichment kits are designed to answer this question. By using combinations of two fluorophores and resistance markers (e.g., GFP:Blast and RFP:Puro), you can carry out dual-enrichment to isolate cells that contain one of each construct, and therefore one KO cassette in each allele
The TrueTag Stem kits include a CMV promoter to drive the expression of the antibiotic resistance marker independently of the endogenous gene promoter. In this way, the enrichment can take place right away and the fluorophore expression will be induced only when the protein of interest is being expressed.
The resistance markers in all TrueTag constructs can be removed using the Cre/Lox system.
The C-terminal is generally recommended to prevent interference with translation of the native protein. In the case of the TrueTag Stem kits, the C-terminal is the only option.
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