Functional verification of U6 promoters from Agaricus bisporus and the feasibility of target gene silencing by shRNA

Abstract

CRISPR/Cas9 system has been proven to be an efficient tool to generate gene mutation. The expression of gRNA guiding Cas9 nuclease to target gene of interest was commonly driven by RNA polymerase III promoter in several organisms, most of which generally took advantage of U6 promoter. For identification of U6 promoter which could guide the expression of gRNA in Agaricus bisporus, we aligned U6 genes retrieved from NCBI against A. bisporus genome. A total of two U6 genes with the typical eukaryotic U6 functional sites and high sequence conservation were obtained. And then shRNA-mediated adenylate cyclase (AC) gene silencing cassette with ∼500 bp upstream of U6 genes was transformed into A. bisporus to explore the function of U6 promoters. Changes in expression of target gene AC and phenotype demonstrated that the two U6 promoters could effectively drive the expression of shRNA to silence the target gene implying that U6 promoters might be capable of driving the expression of gRNA of CRISPR/Cas9 system in A. bisporus. The identification of U6 genes and functional evaluation of U6 promoters would facilitate the application of shRNA and CRISPR/Cas9 system for investigation of function of target gene in A. bisporus in which targeted gene knockouts were likely to be difficult to be implemented because of multinucleate and heterokaryon.