CRISPR Design tool from MIT

[C K]

Dear all,

I want to design 2 guide RNA and create a single base change. Should the target base ideally be located in the middle where the 2 guide RNA bind to my target gene? I'm using the CRISPR design tool from MIT and when I put in a sequence, I receive the error message "Sequence length not within allowed range (23 - 250bp)" Although both sequence types to choose from allow for 23-500bp. Is there a bug in the system?. Any other design tool website that looks for gRNA pairs?
Thank you for your help!

[Xuefeng Zhang - JAX]

Hi there,

Why do you choose to design a pair, rather then one single gRNA, for creating a single base change?

Xuefeng

[Andrew Manon]

@CK

Could you clarify what your goal is? Based on the gRNA pair, you're trying to introduce a targeted single base change via nickase-mediated HDR? 

[C K]

Yes Andrew that is right. Xuefeng, for created a single base change a colleague told me that when using a pair I reduce the rate of  off-targets. I will then introduce a "repair template" for HDR.
When entering 250bp sequence into that program, my target base was not located within a pair of 2 guide RNA. What I did now is I run that program with downstream 200bp sequence and picked a Guide A, and then entered 200bp upstream sequence and picked a Guide B. I will then use those 2 together a a pair.

[Suying Liu]

Hi CK,

Firstly, although it said 23~500bp, it can actually only input 23~250bp. I dont know whether there is an annotation error but it can only input less than 250bp ever since the release of the website (in fact I remember it says 250bp but not 500bp the first time I use the website, maybe the authors are changing the website). 

Secondly, if you are not for therapy purpose, I suggest you use wt cas9 rather than nickase. The HR efficiency is really low unless it's selectable(GFP, resistance etc), so it will be difficiult for you to pick a correct cell clone if you use nickase. Also, off-target is predicted but doesn't have to happen in your clones, and you can always check whether there is off-target in the correct cell clone so it's not a big problem. 

Thirdly, it's important to check whether the gRNA design by crispr.mit.edu is active 'cause this website mainly focus on off-target prediction. Try more website, like CRISPRscan, Bencling, etc and test gRNA activity before you actually use the gRNAs to do your experiment.

Last but not least, when using WT cas9, it's better to have your gRNA clsoer to the point mutation.

Hope that will help.

Suying 

在 2015年12月23日星期三 UTC+8上午10:57:59，C K写道：

[Victor Dillard]

Hi CK,

Check out the knock-in experiment module in the free platform www.deskgen.com - it'll give you both an off- and an on-target analysis for all guides, allow you to designs pairs and single guides, but also design your HR template in just a few clicks :)

- Victor.