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? Thanks!
Um, well yes. Yes. Those are the disadvantages. Also I will add that because the RNA should lead to quicker Cas9 expression, mutagenesis efficiencies will likely be higher than with DNA vectors.
So why use DNA at all? Well, the issues are mostly practical. DNA vectors are easy to customize for CRISPR. Although the issues of efficiency and mosaicism are potentially problematic, I have seen pretty consistent success* in generating simple indel mutations following injections of PX330-style CRISPR-Cas9 DNA plasmids. That is, consistent double digit percentages of founders carrying mutations as assayed by PCR and/or sequencing. In addition, in our core we have obtained HDR-mediated codon editing rates in the 10-20% range using PX330 vectors co-injected with appropriate "donor" oligos. But this is dependent on cooperative CRISPR sites that have a high rate of baseline cleavage.
Another practical consideration is that not everyone can routinely synthesize high-quality RNAs in vitro with consistency. Quality DNA is relatively easy to prepare and QC. RNA is much less so - especially for the 4+ kilobase Cas9 mRNA. OK, so some of you are saying "Come one, my lab makes RNAs all the time - no prob! " . That's awesome, but the empirical observation is that it's not trivial to get proficient at making long mRNAs, and to keep on top of the key reagent issues (RNAses, enzymes going bad, etc.).
Also, CRISPR DNA plasmids are immediately useful for cell culture gene editing experiments. Some labs will be making these anyway so they will have them on hand, ready to go.
What I am also observing - which many others have reported - is that a fraction of CRISPR sites just don't cut very well, even when the sequence characteristics of the site seem OK. (Like, somewhere on the order of 1/3 to 1/4 of CRISPR target sites?) Most of our injections to date have been using DNA plasmids. It's possible that RNAs might save the day for some of these sites.
The ability to do precise HDR-mediated editing/insertions, rather than simple indels, is very compelling and is the direction most of our CRISPR ideas are going in terms of new mouse models. But coding modifications usually have extremely narrow CRISPR target choices that are imposed by the science; if you want to change a codon, you'll probably need a target as close as possible - preferably overlapping the codon. There won't be many to choose from. So getting the highest efficiency cleavage rates may be critical for some of these projects - for these, Cas9 mRNA or protein may be needed.
Finally, these issues of target efficiency really call for pre-validation of sites. This can be done by transfecting CRISPR plasmids into cooperative cell lines, e.g. NIH3T3 for mouse targets, followed by PCR and mismatch cleavage assays, which can then be quantified. But then - if you go through the trouble to do that, you will have generated the DNA plasmids and thus have the DNA reagent ready for injection.
Having said all that, although I really like the convenience of plasmids, the RNA problems are all about sourcing them. A few vendors, such as Sigma-Aldrich can provide custom guide RNAs and Cas9 mRNA that work. (FYI I do not receive any compensation from Sigma). The RNA reagent expense is less than the cost of mouse embryo injections. I suppose zebrafish researchers may balk at the cost, as they will have more capacity to inject fish eggs, in their own labs usually, and may be more willing to make RNAs in-house. For mice, you'll be usually working with a transgenic core and spending thousands of bucks per experiment. Vendor-supplied RNAs may be worth the money. Thanks for the question!
*Actually, "consistent" may be misleading… To clarify, about 75% of the NHEJ projects I've been observing have had this level of success. So - more success than not, but then again, not perfectly consistent.
*Actually, "consistent" may be misleading… To clarify, about 75% of the NHEJ projects I've been observing have had this level of success. So - more success than not, but then again, not perfectly consistent.
Great post thanks Doug.
ReplyDeleteAnother potential reason is that using RNA actually reduces half life of Cas9 in the system. This has the potential benefit of reducing off target cleavage (harder to quantify in vivo), but also on-target cleavage - which if you're trying to knock-in a point mutation on one or both alleles can be important if an NHEJ disruption on the second allele is undesirable.