TITLE: Simple electroporation for efficient CRISPR/Cas9 genome editing in murine zygotes
AUTHORS: Simon E. Tröder, Branko Zevnik

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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 the introduction of specific mutations in C57BL/6N mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement.</div></div>

[STEPS]
?. [guide RNA annealing]
Resuspend lyophilized crRNA, tracrRNA and ssODN in T10E0.1 buffer to 100 µM (e.g. 5 nmol in 50 µl)(Store at -80 °C until use)
?. [guide RNA annealing]
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
?. [guide RNA annealing]
Heat to 95 °C for 5 min and cool down at 5 °C/ min in a thermocycler(crRNA:tracrRNA duplex can be stored for weeks 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
?. [Preparation of the electroporation mix]
Incubate mix at room temperature for 10 min
?. [Preparation of the electroporation mix]
Place tube on ice, add 2 µl ssODN and vortex
?. [Preparation of the electroporation mix]
Quick-spin at 4 °C and keep tube on ice until use
?. [Preparation of the electroporation mix]
Summary of the 20 µl electroporation mix:  ABCD1ReagentStock concentrationFinal concentrationVolume2crRNA:tracrRNA duplex50 µM
&nbsp;4 µM&nbsp;1.6 µl3&nbsp;Cas9 nuclease61 µM (10 µg/µl)&nbsp;4 µM&nbsp;1.3 µl4&nbsp;ssODN&nbsp;100 µM10 µM&nbsp;2.0 µl5&nbsp;Opti-MEM&nbsp;--15.1 µl
ABCD1ReagentStock concentrationFinal concentrationVolume2crRNA:tracrRNA duplex50 µM
&nbsp;4 µM&nbsp;1.6 µl3&nbsp;Cas9 nuclease61 µM (10 µg/µl)&nbsp;4 µM&nbsp;1.3 µl4&nbsp;ssODN&nbsp;100 µM10 µM&nbsp;2.0 µl5&nbsp;Opti-MEM&nbsp;--15.1 µl
?. [Electroporation of zygotes]
Collect zygotes from the oviducts of superovulated females as described in published protocols
?. [Electroporation of zygotes]
Wash the zygotes in five drops of M2
?. [Electroporation of zygotes]
Wash up to 50 zygotes in one drop of Opti-MEM
?. [Electroporation of zygotes]
Transfer zygotes with as little media as possible to the 20 µl electroporation mix
?. [Electroporation of zygotes]
Using a 20 µl pipette transfer the entire drop 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)
?. [Electroporation of zygotes]
Insert cuvette into a standard electroporator (e.g. BioRad Gene Pulser Xcell electroporator)
?. [Electroporation of zygotes]
Apply two square wave pulses at 30 V and 3 ms duration with a 100 ms interval
?. [Electroporation of zygotes]
Retrieve the zygotes by flushing the cuvette with 100 µl pre-incubated M16 using a 100 µl pipette into a culture dish (e.g. 60 mm Center Well Organ Culture Dish)
?. [Electroporation of zygotes]
Wash the cuvette with 100 µl pre-incubated M16
?. [Electroporation of zygotes]
Transfer all zygotes to a new culture dish with 500 µl pre-incubated M16
?. [Electroporation of zygotes]
Incubate zygotes in M16 until the two cell stage and transfer the developed embryos into pseudopregnant foster mice(Embryos may also be transferred at the one cell stage)