DNA or RNA concentrations for mouse embryo #CRISPR injections: some considerations.

I was asked a few questions recently about this subject.  Here's my responses (edited for clarity):

Q: What is the recommended concentration of ssDNA to use for HDR in mouse embryo injections?

This depends if you are doing cytoplasmic (RNA + DNA) or pronuclear (DNA only) injections.  (More on the differences between those injections in the next question.)   Let's consider cytoplasmic injections of guide RNA, cas9 mRNA, and ssDNA as the HDR donor.   Yang et al. (Cell 2013 v154(6)pp.1370-1379) - the first group to publish HDR in mouse embryos -  reported using 50, 100 and 200 ng/ul for these components, respectively, for cytoplasmic injections.     Other groups have apparently reported using lower concentrations for these components with good success.  However, in general the concentrations used for cytoplasmic RNA/DNA injections will be higher than DNA concentrations used for pronuclear injections.   The cytoplasm can tolerate a higher burden of injected nucleic acid than the pronuclei can.

Q:  What are the main differences between cytoplasmic and pronuclear injections?

Cytoplasmic injection is technically slightly easier and (I believe) can result in higher embryo survival and implantation rates, since the risk of damaging the pronucleus is lower.    But it is only appropriate for RNA injections, such as Cas9 mRNA with guide RNAs, with or without HDR DNA donor molecules.     Although Cas9 mRNA injections can be very efficient, they have the disadvantage that they depend critically on the mRNA quality, which can vary depending on who made it, which kit was used, how it was stored and for how long, etc.   I am aware of l difficulties that several groups have encountered with this - some of this is anecdotal but some is from my direct experience. 

Because of this, I prefer injection of DNA plasmids (e.g. PX300) to transiently express Cas9 and guide RNAs.    Although this is slightly less efficient than RNA injections (when the RNA is good), it is very consistent, and it's easy for basically any lab that does cloning to make decent mini prep plasmid DNA suitable for injection.   This is not so straightforward for in vitro mRNA synthesis.       

So, how much DNA can you inject into a pronucleus?   It seems that up to about 10 ng/µl are OK as a maximum concentration, without incurring too much toxicity.    This is a total DNA burden.  A typical HDR experiment will then have at least one guide RNA/cas9 plasmid co-injected with an HDR ssDNA oligo or ds DNA fragment.   How much of each is optimal is still being worked out.    I suggest a 1:1 mass ratio of plasmid concentration to donor DNA concentration.  (note mass ratio, not a molar ratio; e.g. similar ng/ul of plasmid and donor.)

Engineering SNPs in mice with #CRISPR :

Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system.

Authors

Inui M, et al. 

Journal

Sci Rep. 2014 Jun 23;4:5396. doi: 10.1038/srep05396.

Affiliation

#CRISPR #Cas9 protospacer sequence considerations - some current thoughts.

Recent papers prompted me to write about this issue some more - this is building on the April 23 post.

Will your CRISPR target work efficiently?   First, does Cas9 “prefer” any bases within the protospacer sequence?

1. Wang et al, Science 2014, examined a library of protospacers to determine bases that were either generally enriched on Cas9 after loading of the guide RNA, or generally depleted from the cellular guide RNA pool after loading, which in theory should give similar results.  They found (Fig. 3F) that U in positions -4, -2 and -1 (relative to the PAM) lowered Cas9 affinity, and similarly reduced ability to be depleted from the free guide RNA pool.  G’s in these positions had the opposite effect.   C in position -3 helped both loading and depletion of free guide RNAs.  These are trends, but clearly not rules, as simple examination of many published, highly efficient protospacers show obvious exceptions to those data.   Moreover, the trends were not observed in the paper below. 

2. The new paper from Gagnon et al, PLoS ONE 2014, examines bases preferences in slightly different way, by actually sequencing mutations caused by Cas9 cleavage.  This data does not support most of the base preferences as I interpret from Wang et al above, except that a G in the -1 position relative to the PAM (= position #21 in the Gagnon paper Fig 1) had a positive effect on indel generation.   These papers used different systems (cell culture vs fish injections).

Some more important points:

3. GC richness overall is a good thing for protospacer effect.  See Gagnon et al Fig 1B.  This general observation had been reported before, but that’s a nice data figure.  Finally - a reason to be happy that your gene is so GC rich! 

4. More about extending the 5' end of the protospacer:  This one bears repeating.  Ran et all Cell 2014 - one of the first “Nickase” papers - actually tested extending the 5’ sequencing of the protospacer portion of the guide RNA, in hopes it might increase efficiency and/or, crucially, specificity.  That would be nice, wouldn’t it?  Alas, adding more 5’ bases doesn’t seem to have any effect (Fig 1A,B).  But a take-home is this:  Since the RNA Pol III requires G to initiate, simply add a G to the 5’ end of the protospacer if one doesn’t exist already.  In other words, there is no need to choose protospacers that have a G at the 5’ end.   Just add it if it’s not there.   Bam - I just increased your target choices 4 fold (you’re welcome).  Now, truth be told, many target choice search programs don’t require a G in the first position anyway.   But they may not suggest that you add the G, which will enhance transcription of the guide RNA.    

Caveat: while shortening the protospacer 5’ end increases specificity, if you do use that strategy, you do need to select targets with an actual G base at the truncated 5’ end of the protospacer genomic target.  Otherwise - if you just add a G base to the truncated PS - you will be adding it within the spacing of a normal length protospacer, thus creating a mismatch.  This would likely be tolerated at the 5’ end of a 20 base protospacer - as was reported by Gagnon et al - but maybe not for a shorter one - this is yet another thing that needs testing, but would be a useful tweak.  

Recent #CRISPR papers of note: Translocations and deletions in cells, and a review with nice figures from Zhang.

1.  Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system. R. Torres1, M.C. Martin2, A. Garcia1, Juan C. Cigudosa2, J.C. Ramirez1 & S. Rodriguez-Perales2.  Nat Commun. 2014 Jun 3;5:3964. doi: 10.1038/ncomms4964.

 

2. Characterization of Genomic Deletion Efficiency Mediated by CRISPR/Cas9 in Mammalian Cells.  
Canver MC1, Bauer DE2, Dass A2, Yien YY3, Chung J3, Masuda T3, Maeda T3, Paw BH3, Orkin SH4.  J Biol Chem. 2014 Jun 6. pii: jbc.M114.564625. [Epub ahead of print]

3. Development and Applications of CRISPR-Cas9 for Genome Engineering.  (Review). 

Patrick D. Hsu, Eric S. Lander, Feng Zhang.  Cell, Volume 157, Issue 6, p1262–1278, 5 June 2014

Another #CRISPR target identification web tool: CHOPCHOP. http://nar.oxfordjournals.org/content/early/2014/05/26/nar.gku410.long

Just out in Nucleic Acids Research, online May 26.  Nucleic Acids Res. 2014 May 26. pii: gku410. [Epub ahead of print]
CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing.
Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E.