Transfecting Edit-R™ Cas9 mRNA with Synthetic Guide RNA in Cultured Mammalian Cells

Protocol

Transfecting Edit-R™ Cas9 mRNA with Synthetic Guide RNA in Cultured Mammalian Cells

Jul 28, 2021

Abstract

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.

Introduction

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)

Procedure

Time Taken

2 days

Comments

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

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