Monday, June 30, 2014

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.)


  1. January 2015 update: For editing experiments In our transgenic core, we are now advising pronuclear injections with 5 ng/µl for CRISPR plasmid DNA (e.g. PX330) plus 10 ng/µl HDR donor oligo DNA, for a total burden of 15 ng/µl. This seems to be tolerated well in terms of embryo survival.

  2. Hi Douglas, thank you for your comments! I have a question: isn't the disadvantage of injecting DNA the threat of integration and more frequent mosaicism than in the case of RNA as Cas is expressed quicker? Do you have some direct experience with that?