Applications: Genome Editing Using CRISPR/Cas9

CRISPR/Cas9 Genome Editing:
mRNA + gRNA Transfection

TransIT®-mRNA Transfection Kit for mRNA and gRNA Oligonucleotide Delivery

In order to avoid off-target cleavage and unwanted genomic integration of plasmid DNA, Cas9-encoding mRNA can be co-transfected with guide RNA oligonucleotides. Benefits of RNA-based genome editing include:

  • High Specificity Rapid gene expression generates a transient pulse of genome editing activity
  • Ease of Use Deliver mRNA and guide RNA with a single reagent
  • DNA Free No risk of insertional mutagenesis

RNA Transfection Protocol

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CRISPR mRNA and gRNA Delivery Approaches

CRISPR/Cas9 Delivery Methods – Cas9 mRNA + Guide RNA Oligonucleotides. Cas9 is supplied as messenger RNA, and guide RNAs 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.

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Delivery of Cas9 mRNA and CRISPR Guide RNA with TransIT-mRNA Transfection Kit

Efficient Genome Editing with Cas9 mRNA + Guide RNA Oligonucleotides. HEK293T/17, U2OS and NHDF cells were co-transfected with 0.5 µg of Cas9 encoding mRNA, 5meC, ψ (Trilink Biotechnologies) and 25nM of PPIB targeting 2-part gRNA (Dharmacon) using TransIT®-mRNA Transfection Kit (0.5 µl/well of 24-well plate of both mRNA Reagent and Boost, Mirus Bio). A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.

Glossary of CRISPR TermsClick to expand

RNA Transfection Protocol

CRISPR mRNA + 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