CRISPR/Cas9 Genome Editing:
Cas9/gRNA RNP Delivery

TransIT-X2® Dynamic Delivery System and
Ingenio® Electroporation Solution for Ribonucleoprotein (RNP) Delivery

Purified Cas9 Protein can be combined with guide RNA to form an RNP complex to be delivered to cells for rapid and highly efficient genome editing. Benefits of RNP-based genome editing include:

  • High Efficiency Delivery – Deliver Cas9/gRNA complexes to multiple cell types, including hard to transfect cells such as immune and stem cells
  • High Specificity – Pre-formed RNP complexes provide a rapid pulse of genome editing activity
  • DNA Free – No risk of insertional mutagenesis

RNP Delivery Method | Experimental Data | RNP Transfection Protocol


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CRISPR RNP Delivery Approach

CRISPR/Cas9 Delivery Methods – Cas9 RNP. Purified Cas9 protein and guide RNA oligonucleotides are combined to form a ribonucleoprotein (RNP) complex.

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TransIT-X2 Outperforms Lipofectamine for RNP Delivery.

TransIT-X2® Outperforms Lipofectamine® for RNP Delivery. Ribonucleoprotein (RNP) complexes composed of PPIB (cyclophilin B) targeting 2-part gRNA (IDT) and Cas9 protein (PNA Bio) were delivered into HEK293T/17 and U2OS cells using TransIT-X2® Dynamic Delivery System (1 µl/well, Mirus Bio) or Lipofectamine® CRISPRMAX™ (1.5 µl/well and 1 µl/well of Lipofectamine® Cas9 Plus™ Reagent, ThermoFisher) or Lipofectamine® RNAiMAX (1.5 µl/well, ThermoFisher) or Lipofectamine® 3000 (1.5 µl/well and 1 µl/well of P3000™ Reagent, ThermoFisher) in a 24-well format according to the manufacturers’ protocol. Varying levels of gRNA (6 nM or 12 nM) were tested with 6 nM Cas9 protein (PNA Bio). A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.

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CRISPR RNP Delivery Data

Efficient Genome Editing with Cas9 + Guide RNA Ribonucleoprotein Complexes. The RNP complex of PPIB targeting 2-part gRNA (Dharmacon) and Cas9 protein (PNA Bio) was delivered into HEK293T/17, U2OS, NHDF and K562 cells using TransIT-X2® Dynamic Delivery System (1 µl/well of a 24-well plate, Mirus Bio).  A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection. High levels of gene editing can be achieved in cells that were transfected with an RNP complex comprised of 50nM of gRNA and 25nM of Cas9 protein.

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CRISPR RNP Delivery Data in iPSCs

Efficient Genome Editing with Cas9 + Guide RNA Ribonucleoprotein Complexes. TransIT-X2® Dynamic Delivery System was used to deliver Cas9 protein/guide RNA ribonucleoprotein (RNP) complexes in human induced pluripotent stem cells (iPSCs). A T7E1 mismatch assay was used to measure cleavage efficiency at 48 hours post-transfection.

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CRISPR RNP Delivery with Ingenio Electroporation Solution Targeting PPIB & WTAP

CRISPR RNP Delivery with Ingenio® Electroporation Solution. Ribonucleoprotein (RNP) complexes targeting (A) PPIB or (B) WTAP were electroporated into K562 and Jurkat cells. The RNP complex, composed of 750 nM Cas9 protein (EnGen® Cas9 NLS, New England Biolabs) and 1500 nM pre-complexed two-part gRNA (IDT), was electroporated using the Ingenio® Electroporation Solution (Mirus Bio) and a Gene Pulser Xcell™ Eukaryotic System (Bio-Rad® Laboratories). Exponential pulse conditions of 130V (A) & 150V (B), 950 µF for K562 and 150V, 950 µF for Jurkat cells were applied to triplicate 0.2 cm cuvettes, 100 µl volume, 10 x 106 cells/ml +/- RNP complex. A T7E1 mismatch assay was used to measure cleavage efficiency at 48 hours post-transfection. Non-specific bands (NSP) were observed in the negative control of both cell lines. Cleavage efficiency was calculated based on the ratio of cleaved band intensities to the sum of cleaved and uncleaved band intensities minus the average signal of the non-specific band(s) in negative control lanes.

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TransIT-X2 Outperforms Lipofectamine CRISPRMAX for RNP Delivery

TransIT-X2® Outperforms Lipofectamine® CRISPRMAX™ for RNP Delivery. Ribonucleoprotein (RNP) complexes composed of PPIB targeting 2-part gRNA (IDT) and Cas9 protein (PNA Bio) were delivered into HEK293T/17 (A), U2OS (B), and primary H-RPE (C) cells using TransIT-X2® Dynamic Delivery System (1 µl/well, Mirus Bio) or Lipofectamine® CRISPRMAX™ (1.5 µl/well and 1 µl/well of Lipofectamine® Cas9 Plus™ Reagent, ThermoFisher) in a 24-well format according to the manufacturers’ protocol. Varying levels of gRNA (6 nM – 60 nM) were tested with 6 nM Cas9 protein. A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.

 
Glossary of CRISPR TermsClick to expand
 
CRISPR Ribonucleoprotein Transfection ProtocolClick to expand

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Brochure: CRISPR/Cas9 Genome Editing
Poster: Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Cell Engineering
White Paper: Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Cell Engineering