Hello,
We are working on establishing a novel CRISPR-Cas gene-editing system, using Cas genes found in online genome databases. Using artificial gene synthesis and recombinant protein production we have produced novel Cas enzymes.
To test activity and do further work we need guideRNA. Since the guideRNAs are specific to each Cas9 we need to design the RNA molecule from the spacer-, repeat- and tracr sequence. These have been located in the genome. The problem is how to combine them, and if they should be trimmed, and how much.
We would very much appreciate advice from you if you have done this, or have an idea how to do this.
Best from Tromsø,
PhD student Greta Daae Sandsdalen
UiT Arctic Univerity of Norway
Just saw this at the top of the questions section and figured I would toss a quick reeply.
You could first attempt to utilize the closest orthologous scaffold sequences to your novel Cas-enzymes (Wt-tracrRNA:SpCas9 for SpCas9* synthetic). As the wild-type scaffold for SpCas9 associated with the sgRNA has been optimized (you can pull it out of a degenerative sequence screen consistently), like-wise one would imagine there is a multitude of potential sequences that may be able to achieve, for instance DSBs when using novel Cas-enzymes. This may be something that you would consider screening a large number of sequence fragments and test them for activity using an NGS approach. I am working on a similar project, but from the scaffold engineering side to identify novel scaffold variants capable of WT-Cas9 level activity, specificity, etc.
Hi Timothy Thank you for replying to my question. This was very helpful, I will read up on scaffold engineering. Good to know that there is not only one variant that will work. I found a 2012 paper from Doudna and Charpentier's group, describing how they designed their original guide and looking at the genome, where they trimmed the sequences already then. Will also look into the closest orthologous of my novel cas9's.
Thank you for your help. Best, Greta
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