Oct 11, 2018
Version 2
CRISPR Editing of Immortalized Cells with RNPs using Lipofection V.2
- Synthego1,
- Synthego1
- 1Synthego
- Synthego
Protocol Citation: Synthego, Synthego 2018. CRISPR Editing of Immortalized Cells with RNPs using Lipofection. protocols.io https://dx.doi.org/10.17504/protocols.io.uibeuan
Manuscript citation:
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
Synthego uses this protocol and it is working
Created: October 11, 2018
Last Modified: October 11, 2018
Protocol Integer ID: 16675
Keywords: HEK293, A549, U2OS, HeLa, CHO, MCF-7, Lipofectamine, immortalized cell line, CRISPR, transfection, lipofection
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 standard immortalized cell lines (adherent or suspension). Although optimized for HEK293 (human embryonic kidney 293 cells), this protocol is applicable to many other cell lines (A549, U2OS, HeLa, CHO, MCF-7). RNP delivery is accomplished using Lipofectamine™ CRISPRMAX™Transfection Reagent. 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 quality and consistently drive high editing efficiencies. Data that demonstrate editing efficiency in HEK293 cells are included.
Attachments
Guidelines
This protocol is meant to serve as a starting point for lipofection of immortalized cells in a 24-well plate format. It may be necessary to experimentally optimize volumes and ratios for RNP formation for each cell type and for other culture plate formats. It is critical to add reagents in the order recommended in the steps. Prepare the RNP complexes with the LipofectamineTM Cas9 PlusTM Reagent and Opti-MEMTM I Reduced Serum Medium in a separate tube (Tube 1) before adding diluted CRISPRMAXTM Reagent (Tube 2).
Reaction volumes are for EACH WELL and should be scaled up proportionally for the number of wells to be used.
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
All Synthego and CRISPRMAXTM reagents should be stored according to the manufacturer’s recommendations.
This protocol was optimized in HEK293 cells and can be used for other common cell lines such as A549, U2OS, HeLa,
CHO, MCF-7.
Successful transfection is critically dependent on cell density. It may be necessary to optimize cell seeding densities in
order to determine the most appropriate level of confluence for transfection.
For fast growing cells, seed fewer cells. Cell seeding is based on the rate of cell growth. Suggested starting cell numbers
are listed in the protocol below.
In order to maximize CRISPR editing, be sure to include trypsinization (Step 9) in order to perform a reverse transfection of RNPs.
Use cells at lowest passage number possible.
Cas9 nuclease can be diluted in Opti-MEMTM I Reduced Serum Medium in order to achieve a working concentration
according to the plate volume.
Synthego recommends sgRNA:Cas9 ratio of 1.3:1 for RNP formation. It may be necessary to optimize ratios for different cell lines/conditions.
RNP complexes are formed in Opti-MEMTM I Reduced Serum Medium and can be added directly to cells in culture medium irrespective of antibiotics. Following transfection, it is not necessary to remove RNP complexes or to add or
change medium.
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 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.
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
Note: cell seeding may take 1-2 days and incubation after transfection may take 2-3 days.
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
PBS buffer Thermo Fisher ScientificCatalog #10010023
Opti-MEM™ Reduced Serum MediumThermo Fisher ScientificCatalog #31985062
Chemically modified sgRNASynthegoCatalog #Chemically modified sgRNA
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
Cell counterThermo Fisher Scientific
Normal growth medium (Cell-type dependent)
Microcentrifuge tubesEppendorf
Tissue culture platesThermo Fisher Scientific
TrypsinThermo Fisher Scientific
Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (includes Cas9 Plus Reagent and CRISPRMAX Transfection Reagent) Thermo Fisher ScientificCatalog #CMAX00001
Cas9 2NLSSynthegoCatalog #Add at checkout
Safety warnings
Please refer to the SDS (Safety Data Sheet) for safety warnings and hazard information.
Pre-Lipofection - Seed cells
Pre-Lipofection - Seed cells
Seed cells and incubate in 37°C/5% CO2 incubator overnight so that they are 30-70% confluent on the day of transfection (Day 2).
37 °C 5% CO2 incubator
Setup & Lipofection - Assemble RNP Complexes (1.3:1 sgRNA to Cas9 ratio)
Setup & Lipofection - Assemble RNP Complexes (1.3:1 sgRNA to Cas9 ratio)
Dilute sgRNA and Cas9 to 3 μM stock concentrations (3 pmol/μl).
Note
If you followed the Synthego QuickStart Guide, you may have 30 μM stock concentrations (30 pmol/μl), so be sure to dilute further to 3 μM (3 pmol/μl).
Prepare RNPs in microcentrifuge tube (Tube 1). Use the quantities (per reaction) in the table below.
Note
* You may need to experimentally determine the optimum amounts of sgRNA and Cas9 nuclease. Synthego
recommends a ratio of 1.3:1 sgRNA to Cas9 for RNP formation.
Note
Incubate RNPs for 5-10 minutes at room temperature.
00:05:00 Incubation
Setup & Lipofection - Prepare Transfection Solution
Setup & Lipofection - Prepare Transfection Solution
In a separate microcentrifuge tube (Tube 2), dilute Lipofectamine™ CRISPRMAX™ Reagent in Opti-MEM™ I Reduced Serum Medium. Use the quantities (per reaction) in the table below.
25 µL Opti-MEM I Reduced Serum Medium
1.5 µL CRISPRMAX Reagent
Incubate transfection solution for 5 minutes at room temperature.
00:05:00 Incubation
Setup & Lipofection - Make RNP-Transfection Solution
Setup & Lipofection - Make RNP-Transfection Solution
Add the transfection solution (Tube 2) directly to RNPs (Tube 1), and mix well by pipetting up and down.
Incubate for 5-10 minutes at room temperature. Do not exceed 30 minutes.
00:05:00 Incubation
Note
Synthego highly recommends reverse transfection (RNPs are added to wells first and cells are added second), as this method has resulted in high editing efficiencies.
Setup & Lipofection - Prepare Cells
Setup & Lipofection - Prepare Cells
Note
For suspension cells, resuspend in growth medium and mix well. Skip steps 9 and 10 below and proceed to step 11.
Wash cells with 1X PBS (enough to cover bottom of each well), then aspirate PBS.
Add trypsin (enough to cover bottom of each well), incubate for 5 minutes in a humidified 37°C/5% CO2 incubator. Resuspend cells in an equivalent volume of medium to stop the trypsin reaction.
00:05:00 Incubation
Count cells to determine density.
Transfer 0.42 – 1.2 x 105 cells per reaction to a microcentrifuge tube.
Centrifuge cells at 200 x g for 5 minutes.
00:05:00 Centrifugation
Resuspend cells in 500 μl of the growth medium.
500 µL growth medium
Setup & Lipofection - Transfect Cells
Setup & Lipofection - Transfect Cells
Add the RNP-transfection solution mixture to each well of a 24-well tissue culture plate (see table below).
Add cell suspension to each well, and mix by pipetting (see table below).
50 µL RNP-Transfection Solution
500 µL Cell suspension in growth medium
Incubate cells for 2-3 days in a humidified 37°C/5% CO2 incubator.
37 °C Incubation
48:00:00 Incubation
Post-Lipofection - Analysis
Post-Lipofection - Analysis
Extract DNA from cells.
Conduct analyses to determine editing efficiency: PCR, Sanger sequencing, and ICE analysis. 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).
Expected result
Representative Data