CRISPR Nuts and Bolts

Hi All,

Just started learning about CRISPR and would like to design a strategy for gene deletion via NHEJ. I know there is a specific CRISPR worm forum out there on Google, but it looks like no one has posted there in over 6 months so I thought I’d try here before posting to the general CRISPR forum that is not species specific. Still figuring out the nuts and bolts, but would greatly appreciate any answers to questions below. I know it’s quite a few questions, so feel free to tackle any or all of them or suggest references instead (questions 4, 3 and 2, in order of importance). Hopefully, other beginners will find this helpful too. Thanks much!!

  1. What makes something a “C. elegans Cas-9 plasmid?” Just the presence of a C. elegans specific promoter? So if I replace the CBh with an eft-3 in a pX330 (or even use something like a lentiCRISPRv2 with eft-3) - would that make it usable in C. elegans? Are the only commercially available plasmids out there from Addgene? Otherwise, my options are to make my own or ask someone who’s already published?

  2. Is this an okay strategy → inject Cas-9 in a plasmid and separately inject free gRNAs that are not a part of a vector, i.e. rather than having the gRNA be in a plasmid, they will be free floating and just saturate all the cells, so will still be able to guide the Cas-9. Also, since the gRNA cloned into the gRNA plasmid is actually dsDNA, at what point does it become RT into RNA?

  3. Still struggling to understand how to have a reporter that will tell me that the Cas-9 has actually cut my roi and caused a deletion via NHEJ, not just that it entered the cell. The original Church/Calarco paper describes injection of a third plasmid driving expression of mCherry in body-wall muscle and that when they screened for fluorescence they saw a deletion in two of the genes they looked at every time. I don’t understand how injecting a third, independent plasmid reports on having a successful deletion in a different tissue? Even if the mCherry was on the Cas-9 plasmid that would just tell you that the plasmid successfully made it into the cell of interest not that the Cas-9 cut the DNA of your gene of interest and when it was joined up it was in such a way that the mRNA was made truncated/not made. For that to happen wouldn’t you need to introduce donor DNA and a fluorescent marker on the donor DNA plasmid? In short, my question is: what is a good strategy to introduce a fluorescent or better yet phenotypic marker that will report on whether you achieved a deletion within your goi via NHEJ not HR?

  4. I’ve mostly seen vectors using eft-3 or hsp-16.48 as a promoter for Cas-9. My goal is to have Cas-9 directed to the neurons (that where my goi is expressed), so I would want to use unc-119 as the promoter. Does this mean that I need to engineer my own Cas-9 plasmid OR can I use the eft-3 driven Cas-9 plasmid and if I saturate the system with enough Cas-9 and inject at an early enough stage, it will eventually find its way to the neurons given that the gRNAs will be in there (either free floating or driven by U6)? Alternatively, is it possible to add an additional promoter (unc-119) to the eft-3 Cas-9 plasmid?? Or would I have to cut out eft-3 and replace with unc-119? If so, is that the only modification I would have to make within that plasmid to target the Cas-9 to the neurons or would there be any others? (I guess this goes back to my question 1 above of what makes something a C. elegans plasmid)

  5. Just curious - has anyone tried injecting any other tissues besides the gonad and achieved successful genome editing? Or is that too difficult to do technically (i.e. the worms will burst?) How about in mice? Has anyone injected specific tissues and achieved k/o or k/i of a gene?

Hi, I can take a crack at this.

  1. “C. elegans Cas9 plasmid”. For a good description of the various CRISPR systems in C. elegans check out this nice review from Waaijers and Boxem ( Many Ce Cas9 plasmids are codon optimized for worm expression and many have synthetic introns added. You can also decide if you want to inject plasmid DNA or in vitro transcribe and inject mRNA. I know that John Calarco’s (Peft-3), Mike Boxem’s (Peft-3 and Phsp-16.48), and our (Dan Dickinson/Bob Goldstein’s) Cas9 (Peft-3) Cas9 constructs are available on AddGene. I would start with one of these and alter it if you need.

  2. “Free sgRNA?” You can do either. Our plasmid had the sgRNA and Cas9 on the same piece of DNA. Other systems inject separate Cas9 and sgRNA plasmids. Mary-Anne Felix and Michalis Barkoulas have reported injecting PCR products. There is also a report of making a Cas9 line and feeding the sgRNA through the bacteria, though the efficiency in the germline was quite low. One can either inject in in vitro transcribed sgRNA (no need for polyA tailing or 5’ capping) or let the U6 promoter drive it.

  3. “Cas9 activity?” Check out this recent co-CRISPR paper from Craig Mello’s group:
    One issue is that a major failing point for knockin and knockouts is that the guides don’t work. There are rules that predict good guides, but many still fail for reasons that we don’t understand. In the Mello paper they use a mediocre unc-22 guide and found that marker positive animals that gave twitching unc progeny were enriched for mutations or knockins in unrelated genes. They suggest that this strategy selects for animals with good Cas9 expression and good loading of the sgRNA and recommend screening about 20 marker positive animals that give twitching F1 or F2. (screening indels can be done by amplifying the region of interest and analyzing by PAGE, endonucleases like CEL-1 or T7E1, or direct sequencing)

  4. “Promoter choice?” Do you want to delete your gene of interest only in the neurons or in the entire animal? If it’s the former (which would be non-heritable) then you can swap in an unc-119 promoter for eft-3 or hsp-16.48 in a Cas9 vector. I haven’t seen much tissue-specific CRISPR in C. elegans yet. It worked for TALENs though, and the sgRNA feeding paper had better success creating mutations in non-germline tissues (hypodermis I think?) which are non-heritable mutations. If you simply want a mutation in the goi, use an eft-3 and hsp-16.48 driven Cas9, do the co-CRISPR to get a good sgRNA and get homozygotes. You could also consider deleting the entire gene by HR with an unc-119 or drug resistance cassette.

  5. “Other tissues?” I don’t see any point in injecting other tissues. You need to get Cas9 into the cell for transcription/translation. It would be better to use tissue-specific promoters. In mice people mutate ES cells and create transgenics that way.


Thank you very much for the detailed responses. They were very helpful and are greatly appreciated!

On the Co-CRISPR question, I looked at the Mello paper and they do Co-CRISPR with HR rather than NHEJ. NHEJ seems to me the simpler choice. What is the advantage of a deletion via HR over NHEJ? Is it that you leave less to chance with HR and can also do Co-CRISPR vs. not with NHEJ?

The Felix Worm Breeder’s Gazette reference and the Church/Calarco paper do NHEJ, co-inject a reporter plasmid, and the fluorescence if followed through to F2, seems to be a good indicator of the deletion. In both cases a muscle promoter is used. I still don’t understand why GFP marker expression on a totally different plasmid even when the goi has nothing to do with muscle, would be a good predictor of Cas-9 activity in a completely different cell type.

Lastly, do you have any thoughts on eft-3 vs. hsp-16.48 as a Cas-9 promoter?

I’m glad that the responses were helpful!

The Mello paper did both NHEJ (Fig 2) and HR (Figs 3 and 4). If you simply want a mutant, NHEJ is much easier. The efficiencies are often higher. If you need more precision in your mutation (point mutants, loss of a domain, etc.) then HR is still reasonably efficient.

Regarding the fluorescent co-injection markers, most of the papers that use plasmid-based Cas9/sgRNA use the marker to infer a good injection and formation of a robust array. In all cases that I can remember, there is a much greater deletion frequency in marker positive F1 compared to maker negative F1. You are indeed correct that this is not a great predictor of Cas9 activity, which is why the unc-22 co-CRISPR strategy likely yields to improved efficiency.

As to eft-3 vs hsp-16.48 promoter, I prefer the eft-3 as that’s what I’ve largely used. It’s active in a range of tissues (including the germline) and doesn’t require an additional heat shock step. Also, the Calarco plasmid is probably the most widely used thus far. One caveat is that there has been some toxicity from the eft-3 plasmids reported, so a transient Cas9 pulse from a heat shock is an alternative should you have problems with any eft-3 toxicity. Good luck!