A compendium of nonredundant short Polymerase III promoters for CRISPR applications

Abstract Multiplex genome editing via CRISPR is a powerful tool for the simultaneous knockout, activation, and/or repression of distinct genes or noncoding sequences. However, current toolkits for multiplex editing lack diversity. Repeated use of the same promoter in multiple expression cassettes complicates construct assembly and has long been a concern for genetic stability in the host organism. Additionally, using unnecessarily long promoters may increase the genetic load and introduce uncertainties that impact CRISPR efficiency. To address these challenges, we present a collection of short Polymerase III (Pol III) promoters of diverse origins to support increasingly sophisticated genome editing applications in dicots.