I am currently trying to generate CRISPR sgRNA pairs with Sanger Institute's online genome-editing tool. I enter the sequence of interest and it generates hundreds of sgRNA pairs, which I can rank by off-target activity (number of off-target sites with 1-4 mismatches). However, when I check my individual sgRNA sequences with the IDT webtool (link below), it will give many of my sequences low "on-target" scores. Does Sanger (or any other online tool) have a way to incorporate on-target activity into the way it ranks sgRNA pairs?
https://www.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE
I'm usually use CHOPCHOP (https://chopchop.cbu.uib.no/) for gRNA design.
CRISPOR (http://crispor.tefor.net/) and Benchling (https://www.benchling.com/crispr/) also work well.
I have used CCTop , WEG, ChopChop, and IDT and Synthego own platforms. All work fine, just stick to one tool and do not overthink it if you are not interested in the algorithms and beware the number of mismatches allowed in your system. Some sgRNAs I designed had high scores in CCTop and low in ChopChop, yet the actual editing was hight. Even IDT and Synthego platforms, which rely on the same RNP approach, vary in their scores and ranking.
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