Dear Paul,

the design of the sgRNA very much depends on the purpose. Do you want to knockout a GOI, activate or inactivate transcription of a GOI or even perform homology directed repair. There are several specific factors to consider, specific for each purpose.

It is my understanding that most of the published algorithms have only incorporated data generated within the same lab, not elsewhere, which is why I would recommend NOT to use a single tool.

We have done all of the above applications very successfully by following these rules:

1. According to 24463574, truncated sgRNA's improve the specificity and reduce off-target effects. Hence, use 18 or 19 meres.

2. 25184501 has done a great job in figuring out which position of a guide RNA prefers which nucleotide. I have attached Figure 3A. You can simply follow this graph and design the guide yourself.

3. Also, you can find a comprehensive analysis of melting temperatures, GC content, as well as the preferred positions of activating and repressing gRNAs in this nice study: 25307932.

4. If you are using a U6 promoter for the gRNA, try to have the initial G, required for RNA transcription, to be part of the 18-19nt homology.

Good luck. I am certain you'll get it to work.

I used the Zhang Lab genome browser tool for a design in my latest publication, but they have since then released an updated tool.

Both can be found at Zhang Lab: UCSC genome browser tool, mouse

http://www.genome-engineering.org/crispr/?page_id=41

Dear Paul,

the design of the sgRNA very much depends on the purpose. Do you want to knockout a GOI, activate or inactivate transcription of a GOI or even perform homology directed repair. There are several specific factors to consider, specific for each purpose.

It is my understanding that most of the published algorithms have only incorporated data generated within the same lab, not elsewhere, which is why I would recommend NOT to use a single tool.

We have done all of the above applications very successfully by following these rules:

1. According to 24463574, truncated sgRNA's improve the specificity and reduce off-target effects. Hence, use 18 or 19 meres.

2. 25184501 has done a great job in figuring out which position of a guide RNA prefers which nucleotide. I have attached Figure 3A. You can simply follow this graph and design the guide yourself.

3. Also, you can find a comprehensive analysis of melting temperatures, GC content, as well as the preferred positions of activating and repressing gRNAs in this nice study: 25307932.

4. If you are using a U6 promoter for the gRNA, try to have the initial G, required for RNA transcription, to be part of the 18-19nt homology.

Good luck. I am certain you'll get it to work.

Hi Paul, 

I used many tools to design gRNA, but I like these ones  http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx and https://chopchop.rc.fas.harvard.edu/ .

You can use them with different genome and they give you information on the location of your gRNA and also the off-targets.

PS: don't forget to specify your promoter when designing your gRNA.

Good luck.

Hi Paul,

As Manuel said, there isn't (yet) a perfect tool to design sRNAs.

However we have developed CCTop (http://crispr.cos.uni-heidelberg.de/) that, similar to other tools, will give you a ranked list of suggested gRNA based on the predicted (in)efficiency of off-targets. But, different to most of the tools out there, CCTop will give you as well a complete list of the predicted off-targets with some associated properties (mismatches, distance to closest exon, etc) so that you can analyse in depth and make the best selection depending on your aim.

Best,

Juan

I would suggest using CRISPOR (http://crispor.tefor.net/), which is available as a web-based or local Linux-based. This tool allows adding new genome if it is not included in the genome list.

Hi,

I would recommend benchling, it is a wonderful tool, very easy to use.

https://benchling.com/crispr

good luck!

I recommend DeskGen. They have a very intuitive graphical user interface. It is free to use last time I checked.

https://www.deskgen.com

https://sg.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE