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Nano-injection of CRISPR-Cas complexes

By
Maria Milla
on 3/8/19, 11:06 AM 26 views


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. 

MATERIALS

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)

guide RNA

Cas9 protein

Donor DNA (optional)

 

Warning!

Make sure that all solutions (buffers and cell media) have been filtered through a 0.2µm pore before starting the experiment.

PREPARATION

Probe Coating

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.

Complex preparation

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.

Reservoir filling

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.

Cells preparation

In order to facilitate the injection procedure, it is recommended to seed the cells at least 6 hours before the experiment.

Note

 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.

 

PROCEDURE

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.