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


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.

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.

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. 

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.

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

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