TITLE: Cas9 RNP electroporation (suspension and adherent cells)
AUTHORS: Lena Kobel, Eric Aird, Mark Dewitt & Julia Wong

[DESCRIPTION]
This protocol, based on published work, demonstrates how to delivery Cas9 RNP-based gene editing reagents to cultured mamallian cells by electroporation with a Lonza 4d Nucleofector in the small scale 20 ul 16-well strip format. However, it can be upscaled accordingly for the use of the bigger 100 ul cuvettes with the same device.  

Different cell lines need different nucleofector solutions, nucloefector programs and number of cells per reaction. Check https://knowledge.lonza.com/ to find more information about your cell line of interest. 

In addition, consider consulting some of the following papers:
 
1. RNP delivery paper upon which this work is based (Open Access):
https://elifesciences.org/content/3/e04766
2. Paper by an IGI post-doc that details the rationale behind HDR donor design:
https://www.ncbi.nlm.nih.gov/pubmed/26789497

[BEFORE_START]
You will need the following materials:
 
1. Purified Cas9-NLS protein, 40 µM in 1x Cas9 buffer
2. Purified sgRNA from in vitro transcription, >48 µM or synthetic
3. Single-stranded DNA HDR donor, 100 µM (as an IDT Ultramer) (optional)
4. Lonza 4D Nucleofector with X Unit
5. Lonza kit: electroporation solution and 16 reaction small-sized cuvettes (solution specific to your cell type)
6. 1x Cas9 buffer (20 mM HEPES-KOH pH 7.5, 150 mM KCl, 10% glycerol, 1 mM TCEP) and 
    2x Cas9 buffer (40 mM HEPES-KOH pH 7.5, 300 mM KCl, 20% glycerol, 2 mM TCEP)


[STEPS]
SECTION: Prepare RNP Mix
1. 
For a standard reaction, we use 100 pmol Cas9 and 120 pmol sgRNA to form the RNP in a ≤ 5 ul volume. You will need a minimum sgRNA concentration of 48 uM. Mix the following in this order, add Cas9 to the sgRNA slowly while swirling the pipette tip:


 


 

 

 
SECTION: Prepare RNP Mix
2. Allow RNP to form for 10-20 minutes. 
20 min
SECTION: Prepare Cells
3. Count cells. (Trypsinize as needed)
SECTION: Prepare Cells
4. Per reaction, pipette 200'000 cells into a collection tube
SECTION: Prepare Cells
5. Spin 300 x g for 5 minutes to pellet cells softly. While the cells are spinning, prepare a culture plate and cuvette.
 

SECTION: Prepare Cells
6. Prepare a 12-well-plate with 1mL media per well, and pre-warm in the incubator.
 

SECTION: Pre-Nucleofection
7. Prepare and label wells on 20uL nucleofection strips. Configure Lonza 4D using the recommended cell-type program. As an example, for K562 cells it is recommended to use the SF Cell Line kit and program FF-120
  
 
 


 

9. Aspirate the media without disturbing the cell pellet. The pellet is soft so be careful.
SECTION: Nucleofection
12. Add the entire 5 µL RNP mix to the 20 µL cell suspension and mix gently.
SECTION: Nucleofection
13. If an HDR template is used, add it now. For single-stranded donor DNA, add 1uL of 100 uM stock (100 pmoles) to the cell suspension and mix well.
 
SECTION: Nucleofection
14. Transfer 25 µl nucleofection mixes to the multiwell nucleofector strip and cap. Pay attention to the orientation of the cap and cuvette in the nucleofector, which is noted in the manufacturer’s instructions. 
 

SECTION: Nucleofection
15. Insert the cuvette into the nucleofector and select desired well(s) and program(s). Electroporate.
SECTION: Nucleofection
16. Add 80uL of pre-warmed media to each well and then allow cells to sit in nucleofection strips for 10 minutes post-nucleofection. This is supposed to increase efficiency. 
10 min
SECTION: Nucleofection
17. Gently pipette mixture out with a P200 into your pre-warmed 12-well plate. This should get the vast majority of cells, but if you wish, you may wash out the rest with media from the same well with more media.
SECTION: Analysis of Editing
18. Allow cells at least 24 hours to settle and recover before attempted downstream analysis. Consider including non-electroporated controls to test viability. Generally, we check for editing 48-72h after nucleofection using amplicon next generation sequencing combined with CRISPResso2 analysis or using Sanger sequencing combined with an online analysis software (TIDE or ICE from Synthego).
1440 min

For amplicon next generation sequencing of the target site(s), please see:
 

10. Wash the pellet with PBS and repeat the centrifugation
11. Aspirate the PBS without disturbing the cell pellet and resuspend in the 20 ul complete Nucleofector solution
8. Mix the Nucleofector solution and Supplement together for a total of 20 ul per reaction: