Identification, cloning and functional verification of U6 promoter from Cryptococcus neoformans

Abstract:

[Objective] To identify and clone the polymerase Ⅲ U6 promoter from Cryptococcus neoformans (CnU6 promoter), and verify if CnU6 promoter can effectively transcribe shRNA and gRNA of CRISPR/Cas9 system.[Methods] Combining the C. neoformans genome information published in GenBank database and RNA-seq library data from our laboratory, we obtained the U6 RNA sequence with high transcriptional level by bioinformatics analysis. The putative CnU6 promoter was ligated upstream of shRNA and gRNA by EasyGeno and overlapping PCR respectively. Based on shRNA-mediated target gene silence phenotype by RNAi and gene mutation by gRNA-guided Cas9 nuclease mediated target sites editing by CRISPR/Cas9 system, we could identify if CnU6 promoter could drive the transcription of short RNA.[Results] CnU6 promoter could drive the transcribtion of shRNA, which could silence the target gene, and gRNA, which could guide Cas9 nuclease to cut the target site.[Conclusion] The CnU6 promoter from C. neoformans was successfully identified and cloned, which could drive the transcription of shRNA and gRNA efficiently.