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July 3rd, 2025
Version: 2
University of Georgia
plant biology
biorxiv

A compendium of nonredundant short Polymerase III promoters for CRISPR applications

Deguchi, M.Open in Google Scholar•Sinclair, K. M.Open in Google Scholar•Patel, A.Open in Google Scholar•Coile, M.Open in Google Scholar•Ortega, M. A.Open in Google Scholar•Bewg, W. P.Open in Google Scholar•Tsai, C.-J.Open in Google Scholar

Multiplex genome editing via CRISPR is a powerful tool for simultaneous knockout, activation, and/or repression of distinct genes. However, current toolkits for multiplex editing lack diversity. Polymerase III (Pol III) promoters are widely used to express guide RNAs (gRNAs). Repeated sequences, including promoters, in multiple expression cassettes complicate construct assembly and have long been a concern for genetic stability and unwanted silencing. Expressing gRNAs as a polycistronic array eliminates the need for multiple promoters, but these strategies still involve repeated use of tRNAs, ribozymes, or other RNA-cleaving systems, raising additional concerns about variable gRNA processing. Furthermore, using unnecessarily long promoters may increase the genetic load and introduce uncertainties that impact CRISPR efficiency. Here, we present a diverse collection of short Pol III promoters to support increasingly sophisticated genome editing applications in dicots.

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