Simple electroporation for efficient CRISPR/Cas9 genome editing in murine zygotes
Branko Zevnik, Simon E. Tröder
Abstract
Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for highly efficient introduction of specific mutations in mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. This protocol works efficiently with zygotes from a variety of genetic backgrounds and is compatible with other CRISPR nucleases like Cas12a.
Before start
Steps
guide RNA annealing
Resuspend lyophilized crRNA, tracrRNA and ssODN in T10E0.1buffer to 100 µM (e.g., 5 nmol in 50 µl)
(Store at -80 °C until use)
Combine 5 µl crRNA (100 µM) and 5 µl tracrRNA (100 µM) in a nuclease-free PCR tube to yield an equimolar crRNA:tracrRNA duplex solution of 50 µM
Heat to 95 °C for 5 min and cool down at 5 °C/ min in a thermocycler
(crRNA:tracrRNA duplex can be stored for months at -80 °C)
Preparation of the electroporation mix
Add 1.6 µl crRNA:tracrRNA duplex and 1.3 µl Cas9 nuclease to 15.1 µl Opti-MEM in a nuclease-free tube and vortex
(Instead of Cas9 other CRISPR nucleases like Cas12a can be used as well. See Guidelines & Warnings)
Incubate mix at room temperature for 10 min
0h 10m 0s
Place tube on ice, add 2 µl ssODN and vortex
(The ssODN may be left out if desired but must subsequently be compensated by 2 µl Opti-MEM to reach a total electroporation mix of 20 µl)
Quick-spin at 4 °C and keep tube on ice until use
Summary of the 20 µl electroporation mix:
| A | B | C | D |
|---|---|---|---|
| Reagent | Stock concentration | Final concentration | Volume |
| crRNA:tracrRNA duplex | 50 µM | 4 µM | 1.6 µl |
| Cas9 nuclease | 61 µM (10 µg/µl) | 4 µM | 1.3 µl |
| ssODN (optional) | 100 µM | 10 µM | 2.0 µl |
| Opti-MEM | - | - | 15.1 µl |
Electroporation of zygotes
Collect zygotes from the oviducts of superovulated females as described in published protocols
Wash the zygotes in five drops of M2 medium
Wash up to 50 zygotes in one drop of Opti-MEM
(work quickly as zygotes may suffer from extended incubation in Opti-MEM)
Transfer zygotes with as little media as possible to the 20 µl electroporation mix
(e.g., by first transfering the 20 µl electroporation mix onto a culture dish and subsequently adding the zygotes)
Using a 100 µl pipette set to 21 µl volume transfer the entire drop of electroporation mix including the zygotes into a pre-warmed (37 °C) 1 mm electroporation cuvette
(Ensure retrieving all zygotes by quickly aspirating the entire drop. Slow aspiration will leave zygotes behind)
Insert the cuvette into a standard electroporator (e.g., BioRad Gene Pulser Xcell electroporator)
Apply two square wave pulses at 30 V and 3 ms duration with a 100 ms interval
Retrieve the zygotes by flushing the cuvette with 100 µl M2 medium using a 100 µl pipette into a culture dish (e.g. 60 mm Center Well Organ Culture Dish)
Wash the cuvette with 100 µl M2 medium
Transfer all zygotes to a new culture dish containing pre-incubated microdrops of culture medium covered by oil. Wash the zygotes in three drops prior to culture in a fourth drop of culture medium.
(Alternatively, a dish with 500 µl pre-incubated culture medium without oil may be used)
Incubate zygotes in an incubator until the two-cell stage and transfer the developed embryos into pseudopregnant foster mice
(Embryos may also be transferred at the one-cell stage)
