CRISPR/dCas9-mediated transcriptional activation in Aspergillus nidulans

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	CRISPR/dCas9-mediated transcriptional activation in Aspergillus nidulans
	Guo, S.; Parsania, C.; Dong, L.; Wong, K. H.
	2017-03-14
Source Publication	29th Fungal Genetics Conference
Abstract	The well-known and powerful CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat and associated protein Cas9) system has been extensively applied for genome engineering in many organisms. The system can be modified for transcriptional activation or suppression of gene-of-interest by linking a mutated Cas9 that lost the nuclease function (called dCas9) with a transcriptional activation element (e.g. VP16, VP64, VPR and SAM) or a repression element (e.g. KRAB, SID), respectively. In this study, we aim to establish and optimize a CRISPR/dCas9 system for transcriptional activation in A. nidulans. We have generated a strain that expresses the dCas9-VP64 fusion protein and a gRNA expression plasmid carrying an internal fragment of the wA gene for targeting to the wA locus. An efficient strategy for introducing gRNA sequences to the expression plasmid has also been optimized. Similar to the CRISPR/Cas9 system, targeting of dCas9 to specific genomic regions is also mediated by sequence specific gRNAs to PAM sites. However, the location of PAM sites is critical and can greatly influence the degree of transcriptional activation and repression by the CRISPR/dCas9 system. Since many PAM sites can be found across a given promoter region, it is not immediately obvious which one would be ideal for activation/repression. Based on the well-established characteristics (e.g. nucleosome density, chromatin modifications, DNA bendability, sequence conservation, etc.) underlying transcription factors function, we have designed an approach to predict PAM sites suitable for the purpose. As a proof-of- principle, we have tested the CRISPR/dCas9 activation system on genes located within active (e.g. house-keeping genes) or silent heterochromatin (e.g. genes within secondary metabolite clusters) regions in A. nidulans. The success and the level of transcriptional activation were determined using RT-PCR. A “PAM site for activation” prediction program will be developed for public use. We believe that the CRISPR/dCas9 activation system is not only useful for artificial transcriptional activation, but also offer a novel method for activating cryptic secondary metabolite gene clusters in filamentous fungi.
Keyword	CRISPR nidulans
Language	英語English
The Source to Article	PB_Publication
PUB ID	32164
Document Type	Conference paper
Collection	DEPARTMENT OF BIOMEDICAL SCIENCES Faculty of Health Sciences
Corresponding Author	Wong, K. H.
Recommended Citation GB/T 7714	Guo, S.,Parsania, C.,Dong, L.,et al. CRISPR/dCas9-mediated transcriptional activation in Aspergillus nidulans[C], 2017.
APA	Guo, S.., Parsania, C.., Dong, L.., & Wong, K. H. (2017). CRISPR/dCas9-mediated transcriptional activation in Aspergillus nidulans. 29th Fungal Genetics Conference.

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