TrueGuide CRISPR gRNA

Invitrogen TrueGuide Synthetic gRNAs are ready-to-transfect synthetic gRNAs designed and validated to work with the Invitrogen suite of genome editing tools to provide consistent high efficiency editing. Invitrogen is utilizing Synthego's high performance oligo manufacturing to bring you TrueGuide Synthetic gRNAs. Whether you need an economical solution for routine editing tasks or you want to drive maximum editing efficiency, particularly in primary or stem cells, TrueGuide Synthetic gRNAs offer the reagents you require to introduce your specific edit in your cell line.

Predesigned synthetic gRNA for human and mouse knockouts

Incorporate the latest in gRNA design research coupled with our extensive in-house experience. Our proprietary gRNA design algorithms select gRNAs for maximum editing efficiency without compromising specificity.

Invitrogen custom TrueGuide gRNA ordering tool

Whether you have a sequence from a journal article, a colleague, or from your favorite design tool, utilize our simple, fast interface to upload the custom gRNA sequences you would like synthesized.

Build your custom gRNA


Receive your gRNAs in a plate format

Contact us at GEMServices@thermofisher.com

TrueGuide Synthetic gRNA formats

 
TrueGuide 2-piece modified Synthetic gRNA

TrueGuide 1-piece Synthetic gRNA

TrueGuide 1-piece modified Synthetic gRNA
Predesigned knockouts for mouse and human cells      
Custom sequences      
Validated controls available      
Available in plate format      
Ready-to-transfect Annealing step required    
Chemical modifications*      
Ideal for primary and stem cells      

*Chemical modifications include 2´O-Methyl analogs and phosphorothioate linkages to increase editing efficiency and protect against nuclease degradation.

Cell line-specific transfection conditions using Lipofectamine CRISPRMAX Reagent

The following cell line-specific conditions are provided as a starting point for transfecting wild-type cells with TrueGuide Synthetic gRNA and TrueCut Cas9 Protein v2 using the Lipofectamine CRISPRMAX Transfection Reagent. Further optimization of the transfection conditions may be necessary for best results.

NOTE: Scroll to the right within the table to see all data.

Cell type Source Media Cell seeding density/well (×103) one day before transfection TrueCut Cas9 Protein v2/gRNA (ng/pmoles) Lipofectamine Cas9 Plus Reagent/well (μL) Lipofectamine CRISPRMAX Reagent/well (μL)
Well format 96-well 24-well 6-well 96-well 24-well 6-well 96-well 24-well 6-well 96-well 24-well 6-well
HEK293 Human embryonic kidney DMEM 18 90 450 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.4 2 10
U2OS Human osteosarcoma McCoy5A 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
A549 Human epithelial lung carcinoma DMEM 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
THP1 Human peripheral blood monocyte leukemia RPMI 10 50 250 400/2.4 2000/12 10000/60 0.8 4 20 0.3 1.5 7.5
K562* Human leukemia bone marrow RPMI 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
iPSC* Human induced pluripotent stem cells Essential 8 8 40 200 300/2 1500/10 7500/50 0.6 3 15 0.3 1.5 7.5
HepG2 Human hepatocellular carcinoma DMEM 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
MDA- MB231 Human epithelial (breast) adenocarcinoma DMEM 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
N2A Mouse brain neuroblastoma DMEM 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
*Use the Neon Transfection System for higher editing efficiency.

Cell-line specific electroporation conditions using Neon system

The following cell line specific conditions are provided as a starting point for transfecting wild-type cells with TrueGuide Synthetic gRNA and TrueCut Cas9 Protein v2 using the Neon Transfection System 10 μL Kit. Further optimization of the electroporation or nucleofection conditions may be necessary for best results.

Cell type Source Media Number of cells/10-μL reaction (× 103) TrueCut Cas9 Protein v2/gRNA (ng/pmoles) Neon electroporation conditions*
Well format 24-well
HEK293 Human embryonic kidney DMEM 150 1250/7.5 1150 V/20 ms/2 pulses
U2OS Human osteosarcoma McCoy5A 150 1250/7.5 1400 V/15 ms/4 pulses
A549 Human epithelial lung carcinoma DMEM 120 1250/7.5 1200 V/20 ms/4 pulses
THP1 Human peripheral blood monocyte leukemia RPMI 200 2000/12 1700 V/20 ms/1 pulse (#5)
K562 Human leukemia bone marrow RPMI 200 1250/7.5 1700 V/20 ms/1 pulse (#5)
iPSC Human induced pluripotent stem cells Essential 8 80 1500/10 1200 V/20 ms/2 pulses (#14)
iPSC Human induced pluripotent stem cells StemFlex 80 1500/10 1200 V/30 ms/1 pulse (#7)
Human primary T-cell Healthy donor derived OpTmizer + 2% human serum 200 1250/7.5 1600 V/10 ms/3 pulses (#24)
Jurkat T-cell Human peripheral blood lymphocyte RPMI 200 1250/7.5 1700 V/20 ms/1 pulse (#5)
HepG2 Human hepatocellular carcinoma DMEM 120 1250/7.5 1300 V/30 ms/1 pulse (#8)
N2A Mouse brain neuroblastoma DMEM 100 1250/7.5 1400 V/30 ms/1 pulse (#9)
*Recommendations for the Neon electroporation settings are based on the culture conditions tested.

Application data


Up to 90% functional knockout in T-cells

Figure 1. High efficiency functional knockout in T-cells. Human T cells were isolated and activated using Invitrogen Dynabeads magnetic beads (Cat. Nos. 11344D and 11141D, respectively) and then transfected with TrueCut Cas9 Protein v2 and TrueGuide Synthetic gRNA for T cell receptor alpha (TRAC) or beta (TRBC) regions using the Invitrogen Neon delivery system. Following transfection, editing efficiency was measured by (A) the Invitrogen GeneArt Cleavage Detection assay, or by (B,C) functional knockout measured by % T cell receptor negative (TCR–) cells using the Invitrogen Attune NxT Flow Cytometer. Cells analyzed by flow cytometry were stained with T cell receptor–specific antibody conjugated to PE. Here we see up to 90% functional knockout in T cells using our optimized protocol.
 

Genome editing in cancer cells

Figure 2. Prevail with tools to help unravel the complexities of cancer cells. A wide range of cancer cell lines and gene targets were tested to determine editing efficiencies using the modified single TrueGuide Synthetic gRNA (Cat. No. A35511) complexed with TrueCut Cas9 Protein V2 (Cat. No. A36497).  A two-fold increase in efficiency was observed as compared to editing systems from competitors. Here the Cas9 RNP complex was delivered into cell lines indicated using Lipofectamine CRISPRMAX (Cat. No. CMAX00003) and at 72 hours post-transfection, editing efficiency was accessed by Ion Torrent NGS sequencing (NGS) or GeneArt Genomic Cleavage Detection (GCD) Kit (Cat. No. A24372).


Editing efficiencies that outperform the competition

Figure 3. Invitrogen CRISPR genome editing tools provides consistently higher editing efficiency in difficult targets, up to 2 times higher than the competition. Genome editing of multiple gene targets was performed with TrueCut Cas9 Protein V2 and corresponding TrueGuide Synthetic gRNAs. Delivery was achieved using recommended transfection protocols and Lipofectamine CRISPRMAX. The graphs also compare the same experiments using products and recommended protocols another manufacturer. With the Invitrogen system, cleavage efficiency (as measured by %Indel) is improved for low efficient loci (PRKCG-T1 and CMPK1-T1) and shows consistently superior efficiency when compared to competitor products and protocols even for challenging loci.


Cell Viability

Figure 4. Optimized protocols provide minimal cell toxicity. This data set illustrates how the combination of gRNAs synthesized using our proprietary design and delivered using our recommended transfection protocols minimize impact on overall cell viability. Lipofectamine CRISPRMAX were used to complex and deliver TrueCut Cas9 Protein V2 and target specific TrueGuide Synthetic gRNAs for editing several loci including two known to be challenging to edit (PRKCG-T1 and CMPK1-T1). Cell viability was measured by PrestoBlue.

User guide

CRISPR validated protocol collection

Frequently asked questions−TrueGuide Synthetic gRNA

Get your CRISPR guide RNA questions answered

gRNA, or guide RNA, is part of the CRISPR-Cas9 system. The gRNA molecule guides the Cas9 protein to a specific genomic locus via base pairing between the crRNA sequence and the target sequence. The CRISPR-Cas9 system is composed of a short noncoding gRNA that has two components: a target-specific CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA).

sgRNA is the synthetic single guide RNA.

crRNA is the target-specific CRISPR RNA that is part of the gRNA.

tracrRNA is an auxiliary trans-activating crRNA that is needed along with crRNA and Cas9 to form an active Cas9/gRNA RNP complex.

gRNAs are available as chemically synthesized RNA oligos or generated as in vitro transcribed (IVT) gRNAs using the GeneArt Precision gRNA synthesis kit. The IVT gRNA format can also be purchased as a custom ready-to-use option from GEMSErvices@thermofisher.com. Chemically synthesized gRNA are available from Thermo Fisher Scientific in three different formats:

  • Standard format: Invitrogen TrueGuide Synthetic tracrRNA is a 72 nucleotide gRNA scaffold that hybridizes with TrueGuide Synthetic crRNAs to form a gRNA complex that directs Cas9 nuclease to a specific editing site within the genome.  The performance of this two-piece gRNA system is boosted by the addition of 2′ O-Methyl analogs and phosphorothioate internucleotide linkages. These modifications enhance editing efficiency by increasing binding to the target site and inhibiting nuclease degradation, respectively. The modifications are incorporated into the terminal three nucleotides on the 5′ and 3′ ends of both the TrueGuide Synthetic tracrRNA and the TrueGuide Synthetic crRNAs. The TrueGuide Synthetic tracrRNA is available in three package sizes: 5 nmol, 20 nmol, and 100 nmol.
  • Unmodified sgRNA format: Customers can purchase the sgRNA comprised of both the crRNA and tracrRNA as a single long RNA.
  • Modified sgRNA format: Customers can purchase the sgRNA comprised of both the crRNA and tracrRNA as a single long RNA with 2′ O-Methyl and phosphorothioate modifications on both 5′ and 3′ ends of the molecule. 

Chemical modifications include 2’ O-Methyl analogs and phosphorothioate internucleotide linkages at the 5′ and 3′ ends of the molecule. These modifications enhance editing efficiency by increasing binding to the target site and inhibiting nuclease degradation, respectively.

TrueGuide synthetic gRNA along with TrueCut Cas9 Protein V2 provides maximum editing efficiency across broad cell types while minimizing cell toxicity and innate immune responses. Unlike a plasmid-based system, the RNP system is transient with faster turnover. The result is that it minimizes random integrations and off-target events.

TrueGuide Synthetic gRNA sequences are analyzed by mass spectrometry to verify sequence integrity.

To estimate CRISPR/Cas9 mediated editing efficiency in a pooled cell population, one could use GeneArt Cleavage Detection kit (Part No. A24372), perform next generation sequencing (NGS), or use Sanger sequencing-based analysis. Using NGS of the amplicons from edited populations or Sanger sequencing of amplicons cloned into plasmids gives a more accurate estimate of % editing efficiency and indel types.

TrueGuide Synthetic gRNA formats

 
TrueGuide 2-piece modified Synthetic gRNA

TrueGuide 1-piece Synthetic gRNA

TrueGuide 1-piece modified Synthetic gRNA
Predesigned knockouts for mouse and human cells      
Custom sequences      
Validated controls available      
Available in plate format      
Ready-to-transfect Annealing step required    
Chemical modifications*      
Ideal for primary and stem cells      

*Chemical modifications include 2´O-Methyl analogs and phosphorothioate linkages to increase editing efficiency and protect against nuclease degradation.

Cell line-specific transfection conditions using Lipofectamine CRISPRMAX Reagent

The following cell line-specific conditions are provided as a starting point for transfecting wild-type cells with TrueGuide Synthetic gRNA and TrueCut Cas9 Protein v2 using the Lipofectamine CRISPRMAX Transfection Reagent. Further optimization of the transfection conditions may be necessary for best results.

NOTE: Scroll to the right within the table to see all data.

Cell type Source Media Cell seeding density/well (×103) one day before transfection TrueCut Cas9 Protein v2/gRNA (ng/pmoles) Lipofectamine Cas9 Plus Reagent/well (μL) Lipofectamine CRISPRMAX Reagent/well (μL)
Well format 96-well 24-well 6-well 96-well 24-well 6-well 96-well 24-well 6-well 96-well 24-well 6-well
HEK293 Human embryonic kidney DMEM 18 90 450 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.4 2 10
U2OS Human osteosarcoma McCoy5A 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
A549 Human epithelial lung carcinoma DMEM 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
THP1 Human peripheral blood monocyte leukemia RPMI 10 50 250 400/2.4 2000/12 10000/60 0.8 4 20 0.3 1.5 7.5
K562* Human leukemia bone marrow RPMI 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
iPSC* Human induced pluripotent stem cells Essential 8 8 40 200 300/2 1500/10 7500/50 0.6 3 15 0.3 1.5 7.5
HepG2 Human hepatocellular carcinoma DMEM 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
MDA- MB231 Human epithelial (breast) adenocarcinoma DMEM 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
N2A Mouse brain neuroblastoma DMEM 10 50 250 250/1.5 1250/7.5 6250/37.5 0.5 2.5 12.5 0.3 1.5 7.5
*Use the Neon Transfection System for higher editing efficiency.

Cell-line specific electroporation conditions using Neon system

The following cell line specific conditions are provided as a starting point for transfecting wild-type cells with TrueGuide Synthetic gRNA and TrueCut Cas9 Protein v2 using the Neon Transfection System 10 μL Kit. Further optimization of the electroporation or nucleofection conditions may be necessary for best results.

Cell type Source Media Number of cells/10-μL reaction (× 103) TrueCut Cas9 Protein v2/gRNA (ng/pmoles) Neon electroporation conditions*
Well format 24-well
HEK293 Human embryonic kidney DMEM 150 1250/7.5 1150 V/20 ms/2 pulses
U2OS Human osteosarcoma McCoy5A 150 1250/7.5 1400 V/15 ms/4 pulses
A549 Human epithelial lung carcinoma DMEM 120 1250/7.5 1200 V/20 ms/4 pulses
THP1 Human peripheral blood monocyte leukemia RPMI 200 2000/12 1700 V/20 ms/1 pulse (#5)
K562 Human leukemia bone marrow RPMI 200 1250/7.5 1700 V/20 ms/1 pulse (#5)
iPSC Human induced pluripotent stem cells Essential 8 80 1500/10 1200 V/20 ms/2 pulses (#14)
iPSC Human induced pluripotent stem cells StemFlex 80 1500/10 1200 V/30 ms/1 pulse (#7)
Human primary T-cell Healthy donor derived OpTmizer + 2% human serum 200 1250/7.5 1600 V/10 ms/3 pulses (#24)
Jurkat T-cell Human peripheral blood lymphocyte RPMI 200 1250/7.5 1700 V/20 ms/1 pulse (#5)
HepG2 Human hepatocellular carcinoma DMEM 120 1250/7.5 1300 V/30 ms/1 pulse (#8)
N2A Mouse brain neuroblastoma DMEM 100 1250/7.5 1400 V/30 ms/1 pulse (#9)
*Recommendations for the Neon electroporation settings are based on the culture conditions tested.

Application data


Up to 90% functional knockout in T-cells

Figure 1. High efficiency functional knockout in T-cells. Human T cells were isolated and activated using Invitrogen Dynabeads magnetic beads (Cat. Nos. 11344D and 11141D, respectively) and then transfected with TrueCut Cas9 Protein v2 and TrueGuide Synthetic gRNA for T cell receptor alpha (TRAC) or beta (TRBC) regions using the Invitrogen Neon delivery system. Following transfection, editing efficiency was measured by (A) the Invitrogen GeneArt Cleavage Detection assay, or by (B,C) functional knockout measured by % T cell receptor negative (TCR–) cells using the Invitrogen Attune NxT Flow Cytometer. Cells analyzed by flow cytometry were stained with T cell receptor–specific antibody conjugated to PE. Here we see up to 90% functional knockout in T cells using our optimized protocol.
 

Genome editing in cancer cells

Figure 2. Prevail with tools to help unravel the complexities of cancer cells. A wide range of cancer cell lines and gene targets were tested to determine editing efficiencies using the modified single TrueGuide Synthetic gRNA (Cat. No. A35511) complexed with TrueCut Cas9 Protein V2 (Cat. No. A36497).  A two-fold increase in efficiency was observed as compared to editing systems from competitors. Here the Cas9 RNP complex was delivered into cell lines indicated using Lipofectamine CRISPRMAX (Cat. No. CMAX00003) and at 72 hours post-transfection, editing efficiency was accessed by Ion Torrent NGS sequencing (NGS) or GeneArt Genomic Cleavage Detection (GCD) Kit (Cat. No. A24372).


Editing efficiencies that outperform the competition

Figure 3. Invitrogen CRISPR genome editing tools provides consistently higher editing efficiency in difficult targets, up to 2 times higher than the competition. Genome editing of multiple gene targets was performed with TrueCut Cas9 Protein V2 and corresponding TrueGuide Synthetic gRNAs. Delivery was achieved using recommended transfection protocols and Lipofectamine CRISPRMAX. The graphs also compare the same experiments using products and recommended protocols another manufacturer. With the Invitrogen system, cleavage efficiency (as measured by %Indel) is improved for low efficient loci (PRKCG-T1 and CMPK1-T1) and shows consistently superior efficiency when compared to competitor products and protocols even for challenging loci.


Cell Viability

Figure 4. Optimized protocols provide minimal cell toxicity. This data set illustrates how the combination of gRNAs synthesized using our proprietary design and delivered using our recommended transfection protocols minimize impact on overall cell viability. Lipofectamine CRISPRMAX were used to complex and deliver TrueCut Cas9 Protein V2 and target specific TrueGuide Synthetic gRNAs for editing several loci including two known to be challenging to edit (PRKCG-T1 and CMPK1-T1). Cell viability was measured by PrestoBlue.

User guide

CRISPR validated protocol collection

Frequently asked questions−TrueGuide Synthetic gRNA

Get your CRISPR guide RNA questions answered

gRNA, or guide RNA, is part of the CRISPR-Cas9 system. The gRNA molecule guides the Cas9 protein to a specific genomic locus via base pairing between the crRNA sequence and the target sequence. The CRISPR-Cas9 system is composed of a short noncoding gRNA that has two components: a target-specific CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA).

sgRNA is the synthetic single guide RNA.

crRNA is the target-specific CRISPR RNA that is part of the gRNA.

tracrRNA is an auxiliary trans-activating crRNA that is needed along with crRNA and Cas9 to form an active Cas9/gRNA RNP complex.

gRNAs are available as chemically synthesized RNA oligos or generated as in vitro transcribed (IVT) gRNAs using the GeneArt Precision gRNA synthesis kit. The IVT gRNA format can also be purchased as a custom ready-to-use option from GEMSErvices@thermofisher.com. Chemically synthesized gRNA are available from Thermo Fisher Scientific in three different formats:

  • Standard format: Invitrogen TrueGuide Synthetic tracrRNA is a 72 nucleotide gRNA scaffold that hybridizes with TrueGuide Synthetic crRNAs to form a gRNA complex that directs Cas9 nuclease to a specific editing site within the genome.  The performance of this two-piece gRNA system is boosted by the addition of 2′ O-Methyl analogs and phosphorothioate internucleotide linkages. These modifications enhance editing efficiency by increasing binding to the target site and inhibiting nuclease degradation, respectively. The modifications are incorporated into the terminal three nucleotides on the 5′ and 3′ ends of both the TrueGuide Synthetic tracrRNA and the TrueGuide Synthetic crRNAs. The TrueGuide Synthetic tracrRNA is available in three package sizes: 5 nmol, 20 nmol, and 100 nmol.
  • Unmodified sgRNA format: Customers can purchase the sgRNA comprised of both the crRNA and tracrRNA as a single long RNA.
  • Modified sgRNA format: Customers can purchase the sgRNA comprised of both the crRNA and tracrRNA as a single long RNA with 2′ O-Methyl and phosphorothioate modifications on both 5′ and 3′ ends of the molecule. 

Chemical modifications include 2’ O-Methyl analogs and phosphorothioate internucleotide linkages at the 5′ and 3′ ends of the molecule. These modifications enhance editing efficiency by increasing binding to the target site and inhibiting nuclease degradation, respectively.

TrueGuide synthetic gRNA along with TrueCut Cas9 Protein V2 provides maximum editing efficiency across broad cell types while minimizing cell toxicity and innate immune responses. Unlike a plasmid-based system, the RNP system is transient with faster turnover. The result is that it minimizes random integrations and off-target events.

TrueGuide Synthetic gRNA sequences are analyzed by mass spectrometry to verify sequence integrity.

To estimate CRISPR/Cas9 mediated editing efficiency in a pooled cell population, one could use GeneArt Cleavage Detection kit (Part No. A24372), perform next generation sequencing (NGS), or use Sanger sequencing-based analysis. Using NGS of the amplicons from edited populations or Sanger sequencing of amplicons cloned into plasmids gives a more accurate estimate of % editing efficiency and indel types.

Can’t find what you are looking for? Contact us at CRISPR@thermofisher.com

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