Beginner- cannot understand scoring

[Aradhana Mariam Philips]

Hi,
To design the gRNA I gave the input as my Exon of choice from the gene. Proceeding which it leads me to the page where all the possible guides are generated and each guide with their respective off target sites with their own scores. This there are 2 scores mentioned. Which one must I consider? Is there a threshold value I must consider for either score value (that is the guide RNA scores and off target site score)? Please help me with this....

Thank-you

[ania.w...@synthego.com]

Hi Aradhana,

Have you tried our design tool? it makes it relatively easy to design appropriate guides. www.design.synthego.com 

In general, on target scores are not incredibly predictive of how effective a sgRNA is. A score of 90 does not mean it is likely to have a 90% editing efficiency. The ones that are really important are the off target scores. You do not want to use guides that have 1 or 2 mismatches but often 3 mismatches are okay, especially if they are distal to the PAM sequence. Our design tool automatically takes this into account and shows you the top guides based on all of these metrics. 

Hope that helps!

Ania

Field Application Scientist @ Synthego

Genome Engineering Solutions