Assuming there's only 1 Cas9 protein needed to bind each gRNA molecule, we carried out a gel shift assay.
What we don't get is why do we always see a lot of free gRNA migrating in agarose when full complexing should happen? (see attached) The same happens when we keep the gRNA concentration and add more Cas9.
We’ve tried 1:1 ratio of Cas9:gRNA and higher amounts of Cas9 keeping gRNA the same. The RNP band gets more intense but there’s always free gRNA. And we are hybridizing to tracrRNA (Incubation time is 25min at room temperature).
Has anybody tackled this before? or has an explanation for why that might be?
The transfection of plasmids encoding the Cas9 and the guide RNA could lead to undesired permanent recombination and immunogenic responses. We are using a novel strategy which consist in a direct delivery of transient Cas9 protein and single- guide RNA (sgRNA). As other papers did, we do EMSA so we make sure there is a complex formed before delivery.
Here an example:
Article Peptide/Cas9 Nanostructures for Ribonucleoprotein Cell Membr...
Hi Phil Oberacker
!Thanks so much for your recommendations. Actually we followed the same protocol cited in this paper bellow and start with 1:1 ratio used in the paper
https://pubs.rsc.org/en/content/articlelanding/2017/sc/c7sc03918b#!divAbstract
We thought today about decreasing the complexed RNA and start with a ratio of 1:20 of qRNA:Cas9, tomorrow we will run the EMSA and see. We will read your procedure and follow your suggestions.
Regarding your question, we've got the crRNA and ATTO labelled tracrRNA from IDT Technologies. We also use IDT Duplex Buffer and followed manufacture's instructions..
Best
Salam
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