Crispr construct guide or template

[Mega [Andreas Stuermer]]

Hi! 

Am googling like hours for a crispr-construction guide. 

Does anyone have a good paper on this, where you can see how you design it? 

Or even a template plasmid on NCBI where you just paste your target sequence into?? 

Thanks a lot! 

[Mega [Andreas Stuermer]]

As the title says ;)

[Cathal Garvey]

Disclaimer: I've never done this.

shRNA operates on the same principal as microRNA; shRNA is the
human-made version of microRNA, because microRNAs tend to have more than
one use and are structurally complex.

The underlying principal is this: at some stage in eukaryotic evolution,
an anti-viral mechanism evolved that targeted double-stranded RNA,
because eukaryotes generally use single-stranded RNA whereas RNA viruses
usually have a double-stranded stage of their life cycle. This later (I
think?) became co-opted as a final tier of gene expression regulation,
as cells evolved double-stranded "anti-messenger-RNAs" called microRNAs.

The system works by finding double-stranded RNA, and using it as a
recognition template to cut up other RNAs with matching sequences. So if
you want to degrade something with a certain RNA sequence, you'd (in
theory) just make it double-stranded by leaving a spacer (which becomes
a loop) and then adding the inverse of the sequence. The sequence must
be long enough to give the splicing complex room to land on and "hold onto".

There's probably a lot of literature on the specifics of design, like
what flanking nucleotides are best, or which parts of mRNA ought to be
sliced up for maximum efficacy, but the basic idea is you just make
double-stranded RNA to target your unwanted mRNA.

Anyone with more experience and knowledge than that want to comment?

On 21/03/14 16:56, Mega [Andreas Stuermer] wrote:
>
> As the title says ;)
>

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