My recommendations for mixing and diluting #CRISPR gRNAs, mRNAs, and HDR oligo DNAs for mouse zygote injections.

It's super convenient to outsource CRISPR reagent construction to vendors (shout out to my friends at Sigma-Aldrich) and also to outsource generation of CRISPR mice to your friendly neighborhood transgenic core (shout out to the Vanderbilt TMESCSR).   However there still is that step where you may have to actually handle the RNAs/DNAs and mix them together, before handing them off to your friendly transgenic core staff.  I often get asked how I do this.  It's nothing tricky but it does involve RNA, so of course, be clean and careful.  Here are my current recommendations based on receiving RNAs shipped on dry ice from Sigma-Aldrich.

Recommendations for mixing and diluting CRISPR gRNAs , mRNAs, and HDR oligo DNAs for mouse zygote injections.  

Doug Mortlock Feb 2016

•A chart with the recommended final concentrations is found on the TMESCSR website ( https://labnodes.vanderbilt.edu/resource/view/id/11363 ) and is reproduced here.

•Sigma-Aldrich ships the RNAs at slightly higher concentrations than shown below.  Cas9 mRNA is provided at 500 ng/ul and gRNAs at 200 ng/ul.   

•I do not further clean up or process the RNAs in any way prior to dilution.   However, I do carefully re-precipitate the HDR oligos and resuspend them in sterile RNAse-free water.    This can remove some contaminating non-DNA material that is sometimes present in lyophilized oligos as shipped from the vendor.

1.     To prepare “N” injection days worth of injection mixture aliquotes, prepare N+1 tubes as follows.  Use clean, RNAse-free 1.5 ml microfuge tubes, sterile RNAse-free water, and RNAse-free arosol barrier tips.   Pre-rinse each tube by adding 1 ml of the water, vortex, and dump out all the water. Spin down the tubes briefly and remove any lingering dregs of water with a pipette tip. Place all the tubes on ice.

2.     Mix the RNAs, water (and DNA oligo as needed) to create a mix with the correct final concentrations of all reagents and volume that is equal to at least (N x 25) + 5 µl.  Mix briefly by flicking the tube gently (do NOT vortex). 

3.     Spin the mixture at full speed in microfuge for 1 minute. This is to pellet any small particulates – even if none are visible to the eye, before or after this step!

4.     Drawing from the top of the mixture volume, remove 25 µl and aliquot to one of the tubes.  Repeat for each aliquot.  There will be ~5 µl left over.  This hopefully has concentrated any particulate material (that might clog injection needles) and is discarded as a sacrifice to the CRISPR gods.   Although it is NOT usually visibly apparent that there are ANY particulates present, this step is added because it is TYPICAL for the injection technicians to have needle-clogging problems with CRISPR injections.  This reduces embryo survival.   While this pre-spin step may not always solve the problem it may help and is easy to do.

5.     Clearly label the aliquot tubes and bring them on ice to the transgenic core. They should be stored at -20˚ until injection date.   

The chart below was culled from our TMESCSR recommendations as of Feb 2016.  I know 'cause I wrote 'em.

Create the required mixture of components so that it has the final concentrations shown below.

The "min volume to bring to Core" is specific to the Vanderbilt's core's preferences.  So don't use this to argue with your core's staff about what is necessary.  They get to decide that for themselves!

Experiment type	Final conc. Cas9 mRNA	Final conc. all gRNAs	Final conc. all ssDNA donor oligos	Buffer	Min. volume to bring to Core
Knockout experiment, RNA reagents only	100 ng/µl	50 ng/µl	-	Sterile, RNAse-free water	25 µl PER INJECTION DAY
Knock-in experiment, RNA + ssDNA oligo(s)	100 ng/µl	50 ng/µl	200 ng/µl	Sterile, RNAse-free water	25 µl PER INJECTION DAY

Requested deviations from these concentrations will require consultation and approval from the core manager.   The core staff may need to dilute the reagents more if injection problems arise (e.g. clogging).

REQUIREMENTS FOR PURIFICATION:

• We strongly suggest ssDNA oligos be re-precipitated before use to remove potential contaminants from the vendor.    See TMESCSR website for this protocol.