Transfecting Edit-R™ Cas9 mRNA with Synthetic Guide RNA in Cultured Mammalian Cells
- Horizon Team 1
- 1 - Horizon/Perkin Elmer
Jul 28, 2021
This is an abbreviated protocol for transfecting Edit-R™ Cas9 mRNA (Cat #CAS11195, #CAS11859, or #CAS11860) with synthetic guide RNA into cultured mammalian cells using DharmaFECT™ Duo transfection reagent (Cat #T-2010-xx). Synthetic guide RNA can be either synthetic single guide RNA, or synthetic crRNA complexed with tracrRNA. The protocol is intended for use after optimization for your cell line has been completed.
The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that requires a guide RNA for genomic DNA target recognition and generation of DNA double-strand breaks (DSB).
Cas9 nuclease mRNA expresses a human codon-optimized version of the S. pyogenes Cas9 nuclease gene with two nuclear localization signals (NLS). The Cas9 nuclease complexes with a guide RNA for genomic DNA target recognition and cleavage, enabling researchers to perform genome engineering experiments in a completely DNA-free manner.
Cas9 Nuclease mRNA is available in a form that co-expresses either mKate2 or EGFP for transfection optimization or enrichment using fluorescence-activated cell sorting (FACS).
Reagents and Equipment
Edit-R™ Cas9 mRNA (Cat #CAS11195, #CAS11859, or #CAS11860)
DharmaFECT™ Duo transfection reagent (Cat #T-2010-xx)
Time Taken2 days
Erdem Özkaya May 24, 2022
Good day! Can I ask about mRNA production ? Do you use modified nucleotides ? Or pyrophosphatase ? Or which capping strategy are you using ? Cap0, Cap1? Do you purify by HPLC or collagen columns ? Or just normal RNA purification kits? I am trying to establish Base editor mRNA on primary Hematopoietic stem cells but it has it's problems, so I am trying to figure out what might be the solution or what other people are using. I would appreciate any help! Thanks. Best regards, Erdem Özkaya
MediaGEN Protocols Expert Exchanges: Next Generation Techniques to Develop Genetic Medicines
PrimerSelecting a Functional Oligonucleotide from a Random Mix
ProtocolDetergent-Free Nuclei Isolation
ApplicationKnock-out cell lines and their use in signaling pathways
ApplicationBenchling for CRISPR Screening