CRISPR/Cas9 Genome Editing:
pDNA + gRNA Transfection

TransIT-X2® Dynamic Delivery System for Plasmid DNA and Guide RNA Oligonucleotide Delivery

Cas9 protein and guide RNA can both be encoded as plasmid DNA for transfection. Alternatively, Cas9 can be delivered as plasmid DNA, and guide RNA can be supplied as an RNA oligonucleotide. Benefits to these approaches include:

  • Low Cost – Plasmid DNA is a renewable, cost-effective format
  • Flexibility – Cas9 and guide RNA plasmids are suitable for stable or transient transfection
  • Ease of use – Guide RNA oligonucleotide format enables simple retargeting of Cas9 to different loci

DNA/RNA Transfection Protocols


CRISPR Plasmid Delivery Approaches

CRISPR/Cas9 Delivery Methods – Cas9 and Guide RNA Plasmids. (A) Cas9 and guide RNA are encoded on the same plasmid. (B,C) Cas9 and guide RNA(s) are encoded on separate plasmids. (A,B) The wild-type Cas9 enzyme contains two endonuclease domains which cleave the target DNA on both strands when programmed with a guide RNA. (C) The D10A mutation converts Cas9 to a nickase that generates single-stranded breaks in the target DNA. For improved target specificity, Cas9 D10A can be used with paired guide RNAs targeting opposite strands to create staggered double-stranded breaks.

CRISPR Plasmid and gRNA Delivery Approaches

CRISPR/Cas9 Delivery Methods – Cas9 Plasmid + Guide RNA Oligonucleotides. Cas9 is supplied as plasmid DNA, and guide RNA(s) are supplied as either synthetic or in vitro transcribed RNA oligonucleotides. (A) The wild-type Cas9 enzyme contains two endonuclease domains which cleave the target DNA on both strands when programmed with a guide RNA. (B) The D10A mutation converts Cas9 to a nickase that generates single-stranded breaks in the target DNA. For improved target specificity, Cas9 D10A can be used with paired guide RNAs targeting opposite strands to create staggered double-stranded breaks.

High Efficiency CRISPR Genome Editing with TransIT-X2

Efficient Genome Editing with Cas9 Plasmid DNA + Guide RNA Oligonucleotides. HEK293T/17, U2OS and NHDF cells were co-transfected with 0.5 µg of Cas9 encoding pDNA (MilliporeSigma) and 50nM PPIB targeting 2-part gRNA (Dharmacon) using TransIT-X2® Dynamic Delivery System (2 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.

 
Glossary of CRISPR TermsClick to expand
 

Plasmid DNA and Guide RNA Transfection Protocols
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CRISPR pDNA Transfection ProtocolClick to expand
CRISPR pDNA + gRNA 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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