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.

For #CRISPR HDR, use donor oligos that are complementary to the "gRNA strand". A new paper shows why; see my blog post.

(ERRATUM:  I made on correction to this post on March 11 2016.  In the original version of the post I stated that the paper implied that the donor oligo should have "additional length of homology on the PAM-distal side as compared to the PAM-proximal side".  That was a mistake - it turns out the opposite was true.   The authors found that additional length of homology on the PAM-proximal side was favorable.  I got confused because the PAM in figure 3 is on the "bottom" strand, not the top, so the PAM-proximal side is to the left of the cut site in their oligo schematics.  Figure 3 has an "upside down" Cas9 icon in keeping with this fact.  Thank you Scot for letting me know about the error!).


After a long break from blogging... here's a nice nugget of insight about CRISPR-mediated homologous recombination.    Back in late 2014 I blogged about 
Optimal design of ssODNs (donor oligos) for #CRISPR - length and strandedness data? . In that post I pointed out a curious observation that HDR oligos work "better" when they are designed from the strand that is complementary to the protospacer/gRNA sequence.   This was somewhat counterintuitive to me, as one might think that in a complementary HDR oligo would tend to anneal to the gRNA, reducing its availability or kinetics somewhat and generally interfering with Cas9's job.   But empirically, this was not the case; complementary oligos work better.

Now, Richardson et al seem to have found an explanation. (Richardson et al, Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nature Biotechnology (2016, Published online 20 January 2016)). Turns out that following double-stranded DNA cleavage by Cas9, the first component of the DNA molecule that it releases is the 3' end of the DNA strand that corresponds to the protospacer/gRNA.  Quote: Hence, although Cas9 globally dissociates from duplex DNA in a symmetric fashion (Fig. 1c), it appears that the enzyme locally releases the PAM-distal nontarget strand after cleavage but before dissociation."  And here is a nice diagram from the supplemental material of the paper that shows how this strand "breathes" after cleavage.  I thank the senior author, Jacob Corn, for graciously allowing me to reproduce the image here:




(OK - before moving forward, let's get clarity on the terms here; when we're comparing the two strands of the DNA containing the CRISPR target, the "target" strand is the DNA strand that directly will anneal to the gRNA.  Thus, the "target strand" is complementary to the gRNA.  The non-target DNA strand encodes the protospacer and the "NGG" PAM sequence.   Got it? )   

Read the above quote again.  The first bit of DNA that is released by Cas9 is the single-stranded 3' end of the non-target strand.  This immediately suggests 2 things:

1.  Cas9 releases the non-target strand before the target strand.  Thus the non-target strand is available sooner than the target strand to potentially engage with a complementary donor molecule and jump-start homologous recombination.      

2.  The 3' end of the non-target strand, which is "PAM-distal", is released first.  This also suggests that the design considerations for homology might be different for the PAM-distal and PAM-proximal sides of the cleavage location.

For this second point, the practical consideration is that commercially available ssDNA oligos are usually limited to 200 bases or less (depending on the vendor's capabilities; 120 base oligos can work well too).  So, we are limited in the length of homology we can actually apply to each side of the cleavage site.   Instead of centering the oligo (e.g. for a 120 base HDR oligo = ~60 bases of homology on each side) it may be better to skew the oligo design to have more homology on one side of the cut site versus the other.  That is what Richardson et al found - at least for one target they investigated in detail  (See Fig. 3c, d, e.)

Here's another thought. The authors note that Cas9 actually stays on the DNA for quite a while after it cleaves both strands - about 5 hours.    This might have something to do with why DNA repair takes significantly longer on Cas9-cleaved breaks than on breaks induced with radiation - Cas9 may just sitting there, sterically hindering the DNA repair proteins from accessing the free DNA ends at the break.  Perhaps, CRISPR mutagenesis efficiencies could be further enhanced by increasing Cas9's intrinsic off-rate?  On this note, another recent paper by Kleinstiver et al (Keith Joung lab) suggests that directed mutations can destabilize Cas9's non-specific interaction with DNA.   The authors note that this reduces off-target cleavage significantly while preserving on-target cleavage .   While they did not see dramatic increases in on-target efficiency, perhaps in some experimental contexts there might be?    Hmm.