CRISPR-Cas13 equipped with crRNA (complementary to transcripts of interest) can be designed to target ssRNA transcripts in cells.

Upon successful crRNA and ssRNA binding, a fluorescent domain on Cas13 generates a signal, indicating that target has been found.

As far as I know, this method has been used to find one ssRNA target at a time. If we use different wavelength fluorophores for distinct Cas13-crRNA targets, is it theoretically possible to mix them all in the same reaction and distinguish the presence of different targets based on the different fluorescence wavelengths generated during target binding?

In this paper one fluorescently labeled crRNA is used to detect cleavage of one RNA target through complimentary binding. So is it theoretically possible to use several crRNA, each labeled with a different fluorophore, to cleave and detect several target RNAs?

  • $\begingroup$ Your link to the paper is broken, is this what you mean? science.sciencemag.org/content/356/6336/438.abstract. Also note that you talk both about flouresently labeled CRISPR proteinn (easy to do, but always 'on') and labelled crRNA (hard to do, maybe only signal on binding possible). $\endgroup$
    – Nicolai
    Jul 16, 2019 at 11:29
  • $\begingroup$ As far as I understand it's not the crRNA that is fluorescently labeled, but a cleavage reporter that is mixed into the reaction. The cleavage reporter is not even the direct RNA target, but apparently gets "inspecifically" cleaved as a result of "collateral cleavage". So to me it seems this method cannot be used for multiple target detection. Paper also at PMC: ncbi.nlm.nih.gov/sites/ppmc/articles/PMC5526198 $\endgroup$
    – gaspanic
    Nov 3, 2021 at 9:25

2 Answers 2


Answer is no. Because each CRIPSR-Cas(xx) system works as a ribonucleoproteic complex, so they needs to be loaded with a crRNA in order to be working. Functionality is guaranteed while each of the Cas endonuclease domain is associated with a crRNA. You maybe could use multiple Cas13 with multiple crRNA. In molecular diagnosis is common to use orthogonal CRISPR enzymes with different crRNA, as can be seen in this original research.

  • 2
    $\begingroup$ I think your answer is self-contradictory... you said the answer is no but then "maybe could use multiple Cas13 with multiple crRNA", so technically, it's a yes?! My main question was whether it was possible to use multiple crRNA that are labeled with different wavelength fluorophores, so that the signal from target binding could be distinguished based on the color signal. If you look at the paper that I just added in my question, they use one fluorophore to detect cleavage of RNA molecules complimentary to crRNA. What if we use multiple crRNA, each labeled with different fluorophores? $\endgroup$
    – P...
    Jun 16, 2019 at 9:15

It should be possible, I don't see why not. Just as you can use multiple gRNAs, as long as there is an abundance of Cas13 protein you can target multiple sequences at the same time. So if you, let's say, get a green fluorescent probe for RNA 1 and a red fluorescent probe for RNA 2, you'd be able to distinguish their localization.


You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .