Do I need to add an additional 'G' before my 20bp gRNA sequence during cloning (under U6) if the first nucleotide of my gRNA is already a 'G'?

18 October 2024 1 10K Report

Hello, according to published papers: https://www.nature.com/articles/nprot.2013.143/figures/4

It is recommended to have an extra guanine before the 20 bp gRNA sequence, for my efficient transcription from the U6 promoter. If my gRNA already starts with a 'G', do I still need to add an additional guanine?

Yun-Tzai Lee

Mouse U6 promoter transcription starts at the +1 position (23 nt after the TATA box), with G as the preferred initiation nucleotide. See this article:Article Pol III Promoters to Express Small RNAs: Delineation of Tran...

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