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[DESCRIPTION]
This protocol describes how to deliver ribonucleoprotein (RNP) complexes that consist of purified Cas9 nuclease duplexed with chemically modified synthetic single guide RNA (sgRNA) to induced pluripotent stem (iPS) cells derived from human fibroblasts. RNP delivery is accomplished using the Lonza 4D NucleofectorTM unit with 20 μl NucleocuvetteTM strips. Chemically modified sgRNAs are designed to resist exonucleases and innate intracellular immune cascades that can lead to cell death. Although primary cells can be challenging to transfect and manipulate, Synthego chemically modified synthetic sgRNAs are of exceptional purity and consistently drive high editing frequencies.
 
 
    
 Developed by researchers at Ludwig Maximilian University of Munich in conjunction with Synthego
 
 
 

[GUIDELINES]
 Abbreviations:
  
CRISPR: clustered regularly interspaced short palindromic repeats Cas9: CRISPR associated protein 9
sgRNA: single guide RNA
RNP: ribonucleoprotein
 PCR: polymerase chain reaction
ICE: inference of CRISPR edits
FACS: fluorescence-activated cell sorting TE: Tris EDTA
PBS: phosphate-buffered saline
GFP: green fluorescent protein
  
  Important Considerations
  
 Working with RNA and RNPs
  Wearing gloves and using nuclease-free tubes and reagents is recommended in order to avoid RNase contamination.
 Always maintain sterile technique, and use sterile, filter pipette tips.
 All Synthego and NucleofectorTM reagents should be stored according to the manufacturer’s recommendations.
 Synthetic sgRNA should be dissolved in TE buffer and diluted to a working concentration using nuclease-free water. Please consult the Synthego Quick Start Guide for best practices related to dissolving and storing synthetic sgRNAs.
 RNPs can be formed directly in NucleofectorTM solution.
 RNP complexes are stable at room temperature for up to 1 hour (may be stored at 4°C for up to one week, or at -20°C for up to 1 month). Note that RNPs stored at 4°C may become susceptible to contamination from microbial growth after long periods of time.
      
 Suggested Controls
 
 
 
 
    Timeline
  
 
a

  
 Additional Information
  
 For an up-to-date list of all Synthego Protocols and other resources, please visit synthego.com/resources
  
 For technical assistance, contact our Scientific Support Team:
  
 Ph: 888.611.6883
 Email: support@synthego.com
 
 
 
 
 


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SECTION: Setup & Nucleofection - Prepare Destination Plate
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SECTION: Setup & Nucleofection -  Assemble RNP complexes (7.5:1 sgRNA to Cas9 ratio)
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SECTION: Setup & Nucleofection - Prepare Cell/RNP Solution
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SECTION: Setup & Nucleofection - Transfer Cell/RNP Suspension to the Nucleocuvette Strip
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SECTION: Setup & Nucleofection - Transfer Cell/RNP Suspension to the Nucleocuvette Strip
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SECTION: Setup & Nucleofection - Transfect Cells
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SECTION: Setup & Nucleofection - Transfect Cells
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SECTION: Setup & Nucleofection - Add Recovery Medium
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SECTION: Setup & Nucleofection - Plate Cells
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SECTION: Setup & Nucleofection - Plate Cells
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SECTION: Setup & Nucleofection - Plate Cells
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SECTION: Post-Nucleofection - Analysis
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SECTION: Post-Nucleofection - Analysis
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