rules for choosing guides for multiplex CRISPRa and CRISPRi

I was wondering if anybody has information on how to choose guides for optimal gene activation/repression using multiplex CRISPR/Cas9s with only two guides?

I have tried different combinations e.g. two guides with average activity v.s. one poor guide plus an average guide and got mixed results. Sometimes the latter outperformed the former, which is counter-intuitive.

So my question is, how should one choose the two guides for best gene activation/repression? More specifically, how far apart should these two guides be, how far away should them be from the TSS, and what should their individual activity level be for best synergistic results etc?

Any suggestions will be highly appreciated. Many thanks!


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Seashell Griffinabout 3 years ago

Very good question. I think basic ideas are laid down but there are no clear golden rules yet.

Terracotta Fenrirabout 3 years ago

The Genetic Peturbation Platform at the Broad can be used to design guides for i and a (https://www.broadinstitute.org/genetic-perturbation-platform)

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