Mar 30, 2023

Cas9 RNP electroporation (suspension and adherent cells) V.4

Nature Communications
DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw98wgmkj/v4
  • 1ETHZ - ETH Zurich;
  • 2UC Berkeley-IGI;
  • 3ETH
  • CornLab
Jacob E Corn
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw98wgmkj/v4
Protocol Citation: Eric Aird, Mark Dewitt & Julia Wong, Lena Kobel 2023. Cas9 RNP electroporation (suspension and adherent cells). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw98wgmkj/v4Version created by Jacob E Corn
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 02, 2022
Last Modified: March 30, 2023
Protocol  Integer ID: 69481
Keywords: CRISPR, Cas, Cas9, nucleofection, electroporation, genome editing, cas9 rnp electroporation, reagents to cultured mamallian cell, cultured mamallian cell, cell line of interest, different cell line, gene editing, hdr donor design, cas9 rnp, cell line, adherent cell, rationale behind hdr donor design, editing reagent, rnp delivery paper, based gene, cell, lonza 4d nucleofector
Abstract
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
Attachments
Icon for 293T nucleofection protocol from lonza.pdf
293T nucleofection p...
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Materials
STEP MATERIALS
Lonza Nucleofector 4dLonzaCatalog #AAF-1002X
SF Cell Line 4D-Nucleofector® X Kit S (32 RCT)LonzaCatalog #V4XC-2032
Lonza Nucleofector 4dLonzaCatalog #AAF-1002X
SF Cell Line 4D-Nucleofector® X Kit S (32 RCT)LonzaCatalog #V4XC-2032
Protocol materials
SF Cell Line 4D-Nucleofector® X Kit S (32 RCT)LonzaCatalog #V4XC-2032
Lonza Nucleofector 4dLonzaCatalog #AAF-1002X
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)

Prepare RNP Mix

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:


ABCD
StockFinalVolume (ul)
Cas9 buffer2x1x1.3
sgRNA100 uM120 pmol1.2
Cas9 40 uM100 pmol2.5
5


Protocol
NAME
In vitro transcription of guide RNAs and 5'-triphosphate removal
CREATED BY
Jacob E Corn

Note
Optimal RNP formation requires the correct final buffer concentration. If your sgRNA is higher than 100 uM, dilute it with water before forming the RNP. If your sgRNA is as low as 48 uM you can omit the 2xCas9 buffer but this could impact the RNP formation and editing efficiency.

Note
Cas9-NLS is stored in -80°C, sgRNAs are prepped by runoff transcription, Cas9 buffer is kept at 4 °C. During the experiment, keep RNA and protein on ice while not in use.



Allow RNP to form for 10-20 minutes.
00:20:00
Prepare Cells
Count cells. (Trypsinize as needed)
Per reaction, pipette 200'000 cells into a collection tube
Spin 300 x g for 5 minutes to pellet cells softly. While the cells are spinning, prepare a culture plate and cuvette.
Note
Optimal spinning time and g force might vary between cell types

Prepare a 12-well-plate with 1mL media per well, and pre-warm in the incubator.
Note
For different cell types, use the appropriate culture vessel

Pre-Nucleofection
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
Lonza Nucleofector 4dLonzaCatalog #AAF-1002X
SF Cell Line 4D-Nucleofector® X Kit S (32 RCT)LonzaCatalog #V4XC-2032


Note
Commonly used cell types / standard programs:

ABC
Cell typeBufferPulse code
K562SFFF-120
HEK-293TSFDG-130
RPE1P3EA-104
JurkatSECL-120
HCT-116SEEN-113
Stimulated T cellP3EH-115


Mix the Nucleofector solution and Supplement together for a total of 20 ul per reaction:
AB
Volume (ul)
Nucleofector Solution16.4
Supplement3.6
20

Note
Always prepare the mixture fresh because once the Supplement is added, it is stable for only 3 months.

Aspirate the media without disturbing the cell pellet. The pellet is soft so be careful.
Wash the pellet with PBS and repeat the centrifugation
Aspirate the PBS without disturbing the cell pellet and resuspend in the 20 ul complete Nucleofector solution
Nucleofection
Add the entire 5 µL RNP mix to the 20 µL cell suspension and mix gently.
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.
Note
Design the donor to match the guide, according to our NBT paper:
https://www.ncbi.nlm.nih.gov/pubmed/26789497
We order single-stranded donors from IDT, as "Ultramers" and resuspend them to 100 µM final concentration.
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.
Note
Try to not introduce bubbles into the wells of the strip because this might interfere with the electroporation pulse. To remove bubbles, tap the strip on the bench top.

Insert the cuvette into the nucleofector and select desired well(s) and program(s). Electroporate.
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.
00:10:00
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.
Analysis of Editing
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).
24:00:00

For amplicon next generation sequencing of the target site(s), please see:
Protocol
NAME
PCR amplicon next generation sequencing
CREATED BY
Jacob E Corn