Efficient Delivery of Sigma-Aldrich CRISPR via a Non-Liposomal Polymeric Transfection Reagent, TransIT-CRISPR®

Efficient intracellular delivery of CRISPR/Cas9 components is needed to achieve high nuclease activity for genome editing. The potent delivery of CRISPR Cas9 plasmids, RNA and RNP (ribonucleoprotein) Complexes into two adherent cell lines, U2OS and HepaRG was achieved using TransIT-CRISPR.

Product Description

Sigma-Aldrich TransIT-CRISPR is a non-liposomal polymeric transfection reagent for efficient delivery of CRISPR components. TransIT-CRISPR allows for simple, fast and efficient delivery of CRISPR DNA, RNA and ribonucleoprotein (RNP) complexes, composed of Cas9 protein and either in vitro transcribed RNA or synthetic RNA, to various cell types. Also, TransIT-CRISPR effectively delivers siRNA duplexes into various cell lines.

CRISPR/Cas9 Formats:        

  • All in one Sigma-Aldrich CRISPR DNA plasmid
    -  Cas9 protein and guide RNA expressed from the same plasmid
  • Separate Sigma-Aldrich CRISPR DNA plasmids
    -  Cas9 protein and guide RNA expressed from different plasmids
  • SygRNA® crRNA and tracrRNA
  • In vitro transcribed CRISPR RNA

Representation of a CRISPR-Cas9 Ribonucleoprotein (RNP) Complex

Figure 1: Representation of a CRISPR-Cas9 Ribonucleoprotein (RNP) Complex

 

Delivery of Sigma-Aldrich CRISPR/Cas9 reagents using TransIT-CRISPR transfection reagent (Product No. T1706) Click here for product detail page.

CRISPR Plasmid DNA and CRISPR RNP Transfection Protocol in Human Osteosarcoma (U2-OS) cells and a Human Immortalized Cell Line of the Liver (HepaRG)

  • Plate Cells
    • Approximately 18-24 hours before transfection, plate cells in 2.5 ml complete growth medium per well in a 6-well plate. For most cell types, cultures should be > 80% confluent at the time of transfection.
    • For adherent cells: Plate at a density of 0.8 - 3.0 x 105 cells/ml.
    • For U2-OS cells and HepaRG cells: Plate at a density of approximately 1.0 x 105 cells/ml.
    • Incubate cell cultures overnight.
       
  • Prepare TransIT-CRISPR:DNA CRISPR complexes immediately before transfection
    • Warm TransIT-CRISPR to room temperature and vortex gently before using.
    • Place 250 µl of Opti-MEM™ I Reduced Serum Medium in a sterile tube.
    • Add between 2 - 4 µg of (1 µg/µl stock) CRISPR plasmid DNA.
    • Pipet gently to mix completely.
    • Add 3 - 6 µl TransIT-CRISPR (1.5 µl of TransIT-CRISPR per 1 µg of CRISPR plasmid DNA) to the diluted DNA mixture.
    • Pipet gently to mix completely.
    • Incubate at room temperature for 15 - 30 minutes to allow sufficient time for complexes to form.
  • Prepare TransIT-CRISPR:In Vitro Transcribed RNA/Cas9 Protein CRISPR complexes immediately before transfection
    Use between a 1.2 and 5 molar excess of in vitro transcribed RNA to Cas9 protein.
    • Pipet between 1.2 and 5 µg of in vitro transcribed RNA  to a sterile tube on ice.
    • Add between 5 and 10 µg of Cas9 protein to the in vitro transcribed RNA, mix gently and incubate on ice for 30 minutes.
    • Warm TransIT-CRISPR to room temperature and vortex gently before using.
    • Place 250 µl of Opti-MEM™ I Reduced Serum Medium to the Cas9 Ribonucleoprotein (RNP) sterile tube.
    • Add between 5 - 6.25 µl of TransIT-CRISPR to the diluted Cas9 RNP.
    • Pipet gently to mix completely.
    • Incubate at room temperature for 15 - 30 minutes to allow sufficient time for complexes to form.
  • Prepare TransIT-CRISPR:Synthetic RNA/Cas9 Protein CRISPR complexes immediately before transfection
    Use a between a 1 and 5 molar excess of SygRNA® synthetic crRNA & tracrRNA to Cas9 protein.
    • Pipet between 30 and 300 pmol each of synthetic crRNA and tracrRNA into a sterile tube on ice (typically between 1.5 and 15 µl of 20 µM synthetic RNA stock solutions). 
    • Add between 5 and 10 µg of Cas9 protein (30 to 60 pmol) to the synthetic crRNA and tracrRNA, mix gently and incubate on ice for 30 minutes.
    • Warm TransIT-CRISPR to room temperature and vortex gently before using.
    • Place 250 µl of Opti-MEM™ I Reduced Serum Medium to the Cas9 RNP sterile tube.
    • Add between 5 - 6.25 µl of TransIT-CRISPR to the diluted Cas9 RNP.
    • Pipet gently to mix completely.
    • Incubate at room temperature for 15 - 30 minutes to allow sufficient time for complexes to form.
       
  • Distribute the complexes to cells in complete growth medium
    • Add the TransIT-CRISPR:DNA or Cas9 RNP complexes drop - wise to different areas of the wells.
    • Gently rock the culture vessel back - and - forth and from side - to - side to evenly distribute the TransIT-CRISPR:DNA or Cas9 RNP complexes.
    • Incubate for 24-72 hours. It is not necessary to replace the complete growth medium with fresh medium.
    • Harvest cells and assay as required.

Results

Delivery of Sigma-Aldrich CRISPR with Cas9-GFP expression cassettes can be monitored by microscopy and FACS.

Figure 2: Results of immunofluorescence of U2OS osteosarcoma cell lines, which were transfected (using TransIT-CRISPR) with the Sigma-Aldrich CRISPR all-in-one vector co-expressing Cas9+GFP and a gRNA targeting human AAVS1.

 

GFP expression as measured by Flow Cytometry

Figure 3: GFP expression as measured by Flow Cytometry - the histogram on the right had a Sigma-Aldrich CRISPR all-in-one vector co-expressing a gRNA targeting AAVS1 and Cas9+GFP was transfected using TransIT-CRISPR reagent into human U2OS cells. The histogram on the right added Sigma-Aldrich TransIT-CRISPR transfection reagent only.
 

Cel-1 Data Results

Cel-1 assay - detects cutting at the EMX1 site after transfection

Figure 4: Cel-1 assay - detects cutting at the human EMX1 site after transfection with Sigma-Aldrich TransIT-CRISPR transfection reagent with Sigma-Aldrich CRISPR plasmid DNA (A), Cas9 protein-sgRNA (RNP) complexes with in vitro transcribed RNA and synthetic RNA in U2-OS cells (B) and Cas9 protein-sgRNA RNP complexes with in vitro transcribed RNA in HepaRG cells (C). K562 cells, nucleofected with EMX1 in vitro transcribed RNA pre-hybridized with Cas9 protein, Cel-1 assay results are shown in (D) for comparison.
 

Flow Cytometry Results

Tranfection Reagent Flow Cytometry Results

Figure 5: U2-OS cells transfected with CRISPR GFP plasmids, using either Sigma-Aldrich TransIT-CRISPR transfection reagent or a competitor’s transfection reagent, were analyzed for fluorescence via flow cytometry. Results show the percentage of live cells with fluorescence.


Please click here for more information on the Sigma-Aldrich TransIT-CRISPR® transfection reagent (Product No. T1706).

*TransIT-CRISPR is a registered trademark of Mirus Bio LLC.

Materials

     
Related Links