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CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression or activation of gene expression in prokaryotic and eukaryotic cells. It was developed by Lei Stanley Qi and colleagues at the University of California at San Francisco and University of California at Berkeley in 2013. Based on the bacterial genetic immune system - CRISPR (clustered regularly interspaced palindromic repeats) pathway, the technique provides a complementary approach to RNA interference. The difference between CRISPRi and RNAi, though, is that CRISPRi regulates gene expression primarily on the transcriptional level, while RNAi controls genes on the mRNA level. ==Background== Many bacteria and most archaea have an adaptive immune systems which incorporates CRISPR RNA (crRNA) and CRISPR-associated (cas) genes. This minimal CRISPR system has been successfully adapted for generating gene knockouts in many model organisms (bacteria, yeast, fruit flies, zebrafish, mice, humans ). A complementary technology uses catalytically dead Cas9 (usually denoted dead Cas9 or dCas9) lacking endonuclease activity to regulate genes in an RNA-guided manner. Targeting specificity is determined by complementary base-pairing of a single guide RNA (sgRNA) to the genomic loci. sgRNA is a chimeric noncoding RNA that can be subdivided into three regions: a 20 nt base-pairing sequence, a 42 nt dCas9-binding hairpin and a 40 nt terminator. When designing a synthetic sgRNA, only the 20 nt base-pairing sequence is modified from the overall template. Additionally, secondary variables must be considered: off-target effects (for which a simple BLAST run of the base-pairing sequence is required), maintenance of the dCas9-binding hairpin structure and to ensure no restriction sites are present in the new sgRNA as this may pose a problem in downstream cloning steps. Due to the simplicity of sgRNA design, this technology is amenable to genome-wide scaling. CRISPRi relies on the generation of catalytically inactive Cas9. This is accomplished by introducing point mutations in the two catalytic residues (D10A and H840A) of the gene encoding Cas9. In doing so, dCas9 is unable to cleave dsDNA but retains the ability to target DNA. Taken together sgRNA and dCas9 provide a minimum system for gene-specific regulation in any organism. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「CRISPR interference」の詳細全文を読む スポンサード リンク
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