Abstract
CRISPR-Cas9 system uses a guide RNA which functions in conjunction with Cas9 proteins to target a DNA and cleaves double-strand DNA. This phenomenon raises a question whether an artificial small RNA (asRNA), composed of a Dicer–binding RNA element and an antisense RNA, could also be used to induce Dicer to process and degrade a specific RNA. If so, we could develop a new method which is named DICERi for gene silencing or RNA editing. To prove the feasibility of asRNA, we selected MALAT-1 as target and used Hela and MDA-MB-231 cells as experimental models. The results of qRT-PCR showed that the introduction of asRNA decreased the relative expression level of target gene significantly. Next, we analyzed cell proliferation using CCK-8 and EdU staining assays, and then cell migration using wound scratch and Transwell invasion assays. We found that cell proliferation and cell migration were both suppressed remarkably after asRNA was expressed in Hela and MDA-MB-231 cells. Cell apoptosis was also detected through Hoechst staining and ELISA assays and the data indicated that he numbers of apoptotic cell in experimental groups significantly increased compared with negative controls. In order to prove that the gene silencing effects were caused by Dicer, we co-transfected shRNA silencing Dicer and asRNA. The relative expression levels of Dicer and MALAT-1 were both detected and the results indicated that when the cleavage role of Dicer was silenced, the relative expression level of MALAT-1 was not affected after the introduction of asRNA. All the above results demonstrated that these devices directed by Dicer effectively excised target RNA and repressed the target genes, thus causing phenotypic changes. Our works adds a new dimension to gene regulating technologies and may have broad applications in construction of gene circuits.
Highlights
As adaptive immune defenses of bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have become a general and powerful tool for genome editing, especially the type II bacterial CRISPR/Cas9 system [1,2,3,4]
The relative expression levels of Dicer and metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) were both detected and the results indicated that when the cleavage role of Dicer was silenced, the relative expression level of MALAT-1 was not affected after the introduction of artificial small RNA (asRNA)
We used an oligomer RNA [26](Supplementary Table S1) which had a great affinity for Dicer and added it to an antisense RNA targeting MALAT-1 RNA to form an artificial small RNA (Supplementary Table S2) (Figure 1)
Summary
As adaptive immune defenses of bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have become a general and powerful tool for genome editing, especially the type II bacterial CRISPR/Cas system [1,2,3,4]. The “spacer” sequences match the genomes www.impactjournals.com/oncotarget of bacteriophages and other mobile genetics elements [57]. The repeat spacer array is transcribed and processed to generate a small crRNA to recognize the target sequences [8,9,10,11,12]. The element flanking the repeat spacer array is the CRISPR-associated (cas) gene encoding the Cas, a double-stranded DNA endonuclease that employs the crRNA to guide the cleavage of target site [13]. Scientists have successfully fused the crRNA and tracrRNA to generate a small guide RNA to simplify the system [16]
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