Nano-injection of CRISPR-Cas complexes
Nano-injection provides a quick and efficient solution for the delivery of the CRISPR-Cas complexes (RNP complexes) into a specific cell compartment. This protocol provides information for an efficient injection of RNP complexes into HeLa nucleus using the FluidFM® technology.
FluidFM® nanosyringe, 800nm aperture, 2N/m nominal stiffness
TE buffer (10mM Tris-HCl pH7.4, 0.25mM EDTA)
HeLa cells growing in a TC treated 6-well plate
CO2 independent growth medium, supplemented with 2nM L-Glutamine, 10% FBS and penicillin/streptomycin
Centrifugal filter (e.g. Ultrafree-MC VV centrifugal filters from Millipore)
Donor DNA (optional)
Make sure that all solutions (buffers and cell media) have been filtered through a 0.2µm pore before starting the experiment.
The surface of the FluidFM® nanosyringe needs to be hydrophobic to avoid cell adhesion. Therefore, a thin layer of a silicone based solution (Sigmacote®) is added to the probe, following the coating protocol. The probe can be coated and stored up to one week before its use.
Dilute 2µg of guide RNA in 80µl of TE buffer.
Add 2µg of Cas9 protein.
Add donor DNA (final concentration between 5 and 20 ng/µl).
Incubate at room temperature 15 min to allow the assembly of RNP complexes.
Filter the solution through a centrifugal filter.
Dispense 1µl of the RNP complex solution into the probe reservoir according to the probe preparation protocol. In order to avoid the drying of the solution into the probe, it is strongly recommended to load the probe on the BOT as fast as possible after this step.
In order to facilitate the injection procedure, it is recommended to seed the cells at least 6 hours before the experiment.
Using medium without phenol red helps to obtain a better visualization of the injection of fluorescent compounds. Therefore, follow the following steps:
Just before the injection procedure, remove the cell culture medium.
Add 2 ml of CO2 independent growth medium supplemented with 2nM of L-Glutamine, 10% FBS and penicillin/streptomycin.
Prepare the system
Turn on the BOT, the microscope and the computer.
Initialize the software.
Load the probe into the probe holder in the right port of the BOT.
Load the TC-treated 6-well plate containing the cells in the left port of the BOT.
Run the Prepare System workflow.
Once the system is ready, run the Injection workflow.
Selection of the cells
Use one of the proposed tools to target the nucleus or the cytoplasm of the desired cell. When performing cytoplasm injection, it is strongly recommended to select a point close to the nucleus. Otherwise, the probe can not enter properly into the cell and the product would be released in the cell culture medium.
Set parameters for injection
Here, CRISPR-Cas complexes are injected into HeLa cells nucleus. For this protocol, the parameters used should be the following:
Parameter Value Setpoint 1µN Approach Speed 50µm/s Retraction Speed 5µm/s Pressure 75mbar Injection Time 3s Retraction Distance
Optimization of the parameters needs to be adjusted when using other cell lines or culture dishes.
HeLa cells injected with RNP complexes. The tracRNA is labelled with Atto 550 (IDT®).