"Another important concern of the CRISPR/Cas system as an antiviral agent is its specificity . The recognition sequence 5′-N20NGG-3′ of the CRISPR/Cas system is relatively short and mismatches at its 5′-end are tolerable , making it prone to induce high off-target effects . In our study, Cas9 induced apoptosis and cellular proliferation inhibition were observed only in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells, suggesting, at least to some extent, that the CRISPR/Cas system was specific enough to distinguish HPV positive cells from HPV negative cells. "
[Show abstract][Hide abstract] ABSTRACT: High-risk human papillomavirus (HR-HPV) has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.
BioMed Research International 07/2014; 2014(3):612823. DOI:10.1155/2014/612823 · 3.17 Impact Factor
"RNA pol III promoters, such as U6 and H1, are commonly used to express these small RNAs. It is believed that mouse U6 promoter transcription starts at the +1 position (23 nt after the TATA box), with G as the preferred initiation nucleotide.23,24,25 However, the exact U6 transcription initiation site has not been rigorously studied. "
[Show abstract][Hide abstract] ABSTRACT: Pol III promoters such as U6 are commonly used to express small RNAs, including small interfering RNA, short hairpin RNA, and guide RNA, for the clustered regularly interspaced short palindromic repeats genome-editing system. However, whether the small RNAs were precisely expressed as desired has not been studied. Here, using deep sequencing to analyze small RNAs, we show that, for mouse U6 promoter, sequences immediately upstream of the putative initiation site, which is often modified to accommodate the restriction enzyme sites that enable easy cloning of small RNAs, are critical for precise transcription initiation. When the promoter is kept unmodified, transcription starts precisely from the first available A or G within the range of positions -1 to +2. In addition, we show that transcription from another commonly used pol III promoter, H1, starts at multiple sites, which results in variability at the 5' end of the transcripts. Thus, inaccuracy of 5' end of small RNA transcripts might be a common problem when using these promoters to express small RNAs based on currently believed concepts. Our study provides general guidelines for minimizing the variability of initiation, thereby enabling more accurate expression of small RNAs.
"However, RGENs still pose several unanswered questions for research applications (Mussolino and Cathomen, 2013). It remains unclear whether the selection of the target sequence is crucial to achieve effective targeted cleavage. "
[Show abstract][Hide abstract] ABSTRACT: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5' end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka.
Biology Open 04/2014; 3(5). DOI:10.1242/bio.20148177 · 2.42 Impact Factor
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