Oct 04, 2018
CRISPR Editing of Immortalized Cell Lines with RNPs using Nucleofection
- Synthego1,
- Synthego1
- 1Synthego
- Synthego
Protocol Citation: Synthego, Synthego 2018. CRISPR Editing of Immortalized Cell Lines with RNPs using Nucleofection. protocols.io https://dx.doi.org/10.17504/protocols.io.spnedme
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 at Synthego and it is working
Created: August 16, 2018
Last Modified: October 04, 2018
Protocol Integer ID: 14798
Keywords: CRISPR, Cas9, Synthego
Abstract
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 immortalized adherent cells or suspension cells. RNP delivery is accomplished using the Lonza 4D NucleofectorTM unit with 16-well NucleocuvetteTM Strips. A option for knock-in is included. Chemically modified sgRNAs are designed to resist exonucleases and innate intracellular immune cascades that can lead to cell death. Synthego chemically modified synthetic sgRNAs are of exceptional purity and consistently drive high editing efficiencies.
Attachments
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 ssODNs: single-stranded donor oligonucleotides HDR: homology-directed repair
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
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
Materials
MATERIALS
Chemically modified sgRNASynthegoCatalog #Chemically modified sgRNA
Cas9 2NLS nuclease (S. pyogenes)SynthegoCatalog #Cas9 2NLS nuclease
Positive control (optional); Recommended: human RELA sgRNA, CDC42BPB sgRNASynthego
Transfection control (optional); Recommended: pMAX GFP (Lonza), GFP mRNA (SBI)
TE buffer (Included with Synthego sgRNA)Synthego
Nuclease-free waterThermo Fisher ScientificCatalog #R0581
4D-Nucleofector System with X UnitLonzaCatalog #AAF-1002X
4D-Nucleofector® X Kit S (32 RCT) specific for cell typeLonzaCatalog #V4XC-1032
Cell counterThermo Fisher Scientific
Normal growth medium (Cell-type dependent)
TrypLE Express or preferred cell dissociation reagentThermo Fisher Scientific
1X PBS, cell culture gradeThermo Fisher Scientific
12-well tissue culture platesCorning
Microcentrifuge tubesEppendorf
ssDNA HDR template (optional)
Safety warnings
Please refer to the SDS (Safety Data Sheet) for safety warnings and hazard information.
Pre-Nucleofection - Seed Cells
Pre-Nucleofection - Seed Cells
Subculture cells 2 days before nucleofection and seed cells in an appropriately sized vessel so that they are 70-80% confluent on the day of transfection. Each nucleofection reaction will require ~1.5 x 105 cells.
Note
Culturing cells for additional days may be necessary to reach the desired confluency.
48:00:00 Subculturing cells
Setup & Nucleofection - Prepare Destination Plate
Setup & Nucleofection - Prepare Destination Plate
Pre-warm 1 ml of normal growth medium in each well of a 12-well cell culture plate per reaction.
1 mL normal growth medium
Note
This will serve as the destination plate after nucleofection.
Setup & Nucleofection - Assemble RNP Complexes (9:1 sgRNA to Cas9 ratio)
Setup & Nucleofection - Assemble RNP Complexes (9:1 sgRNA to Cas9 ratio)
In appropriate plates/tubes, assemble RNP complexes in the order shown below. Synthego recommends sgRNA:Cas9 ratios between 3:1 and 9:1 for RNP formation. Below is an example experiment using a sgRNA:Cas9 ratio of 9:1.
Note
Knock-in Option: to knock in small inserts (<50 bp), an ssDNA HDR Template can be added. The recommended length of each homology arm is at least 50 bp. Add 1 µl 60 µM ssDNA HDR Template per reaction to each well. Optimization may be required. To knock in larger inserts and for more information on designing knock-in experiments, see Tips and Tricks: Design and Optimization of CRISPR Knock-in Experiments.
Incubate RNPs for 10 minutes at room temperature.
Setup & Nucleofection - Prepare Cell Suspension
Setup & Nucleofection - Prepare Cell Suspension
Note
For suspension cells: spin down cells before each aspiration of culture medium and washes (step 5). Skip
steps 6 and 7 below.
Aspirate cell culture medium and wash cells 1-2 times with appropriate volume of 1X concentration of PBS.
Note
Do not shake or hit the flask to dislodge cells, as this may lead to clumping and inaccuracies in cell counting.
Setup & Nucleofection - Prepare Cells
Setup & Nucleofection - Prepare Cells
Add appropriate amount of TrypLE Express and incubate the cells for ~5 minutes, or until they detach from the plate completely.
00:05:00 Incubation
Setup & Nucleofection - Prepare Cell Suspension
Setup & Nucleofection - Prepare Cell Suspension
Neutralize the dissociation reaction with at least 2X volume of normal growth medium.
Count the cells to determine the cell density.
Aliquot enough cells to have 1.5 x 105 cells/reaction.
Centrifuge cells at 90 x g for 8-10 minutes at room temperature.
Note
The cell pellets will not be packed tightly, so care is required when removing the supernatant.
00:08:00 Centrifugation
Setup & Nucleofection - Prepare Cell/RNP Solution
Setup & Nucleofection - Prepare Cell/RNP Solution
Resuspend the cell pellet in 5 μl NucleofectorTM Solution per reaction.
Note
Work quickly, but carefully, and avoid leaving cells in the NucleofectorTM Solution for longer than 15 minutes. Avoid bubble formation.
5 µL Nucleofector Solution
Add 5 μl of cell suspension to 25 μl of RNP solution to make 30 μl of cell-RNP solution per reaction.
5 µL cell suspension
25 µL RNP solution
Setup & Nucleofection - Transfer Cell/RNP Solution to the Nucleocuvette Strip
Setup & Nucleofection - Transfer Cell/RNP Solution to the Nucleocuvette Strip
For each reaction, transfer all 30 μl of cell-RNP solution to a well of the NucleocuvetteTM strip and click the lid into place.
Gently tap the NucleocuvetteTM strip on the benchtop to make sure that each sample covers the bottom of each well and that there are no bubbles in the wells.
Setup & Nucleofection - Transfect Cells
Setup & Nucleofection - Transfect Cells
Pre-program the NucleofectorTM depending on the cell type per reaction.
Note
Make sure that the entire Nucleofector Supplement is added to the Nucleofector Solution (according to manufacturer's protocol) and that the mixture is not more than 3 months old.
Place the NucleocuvetteTM strip with closed lid into the retainer of the 4D-X Core unit. Check for proper orientation of the NucleocuvetteTM strip. Larger cutout is the top (A1 and A2) and smaller cutout is the bottom (H1 and H2).
Press “Start” on the display of the core unit. After run completion, the screen should display a green “+” over the wells that were successfully transfected. Remove the cuvette strips from the Core unit.
Note
Some cell types require a 10-minute incubation at room temperature after nucleofection. Please consult the optimized Lonza protocol to see if this is a necessary step for your cell line.
Setup & Nucleofection - Add Recovery Medium
Setup & Nucleofection - Add Recovery Medium
Carefully resuspend the cells in each well of the NucleocuvetteTM strip with 70 μl of pre-warmed growth medium, and mix gently by pipetting up and down 2-3 times.
70 µL pre-warmed growth medium
Setup & Nucleofection - Plate Cells
Setup & Nucleofection - Plate Cells
Transfer all 100 μl to the pre-warmed 12-well tissue culture plate (prepared in step 2)
Incubate the cells for 2-3 days in a humidified 37°C/5% CO2 incubator.
37 °C Incubation
48:00:00 Incubation
Post-Nucleofection - Analysis
Post-Nucleofection - Analysis
Extract DNA from cells 48 hours after transfection.
Conduct analyses to determine editing efficiency: PCR, Sanger sequencing, and
. Next-Gen Sequencing, FACS, or functional tests may be conducted as alternatives.
Note
Option: If storing cells for future use is desired, split cells into two groups (one for analysis and one for cell culture).