Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression.

Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA
Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022. PubMed Article

We have repurposed the bacterial immune system, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) pathway, as an RNA-guided DNA binding platform, to repress expression of arbitrary genes in bacteria or human cells (Qi et al.). This CRISPR interfering system (CRISPRi), works independently of host cellular machineries, requiring only a nuclease-deficient Cas9 (dCas9) protein and a customized single guide RNA (sgRNA) designed with a 20 bp complementary region to any gene of interest. Co-expression of dCas9 and sgRNA can efficiently block transcription (in bacteria, ~300-fold repression) by interfering with transcriptional elongation, RNA polymerase binding, or transcription factor binding.

The binding specificity is determined jointly by 20 bp matching region on the sgRNA and a short DNA motif (protospacer adjacent motif or PAM, sequence: NGG) juxtaposed to the DNA complementary region. The uniqueness of CRISPRi, as compared to several recently published works on applying the wild-type CRISPR system for genome mutagenesis (Cong et al., Mali et al., Jiang et al., Hwang et al.), is that the nuclease-deficient mutant could silence transcription on the gene expression level without genetically altering the target loci. Thus, CRISPRi is a system that can regulate a genome instead of modifying a genome.

Two-plasmid CRISPRi system for bacterial gene knockdown

The first plasmid (pdCas9_bacteria) contains an anhydrotetracycline (aTc)-inducible dCas9 gene on a p15A vector with chloramphenicol resistance. The second plasmid (pgRNA_bacteria ) contains a constitutive sgRNA expression cassette on a ColE1 vector with ampicillin resistance, wherein the N20 can be custom designed to target arbitrary sequences in the genome. Co-expression of both plasmids in bacteria could cause up to 300-fold repress on targeted genes.

Also see, two-plasmid CRISPRi system for mammalian gene knockdown.

Plasmids from Article

ID	Plasmid	Purpose
44246	pdCas9-humanized	Expression of a catalytically inactive, human codon-optimized Cas9 under the control of Murine Stem Cell retroVirus LTR promoter for mammalian gene knockdown
44247	pdCas9::BFP-humanized	Expression of a catalytically inactive, human codon-optimized Cas9-BFP fusion under the control of Murine Stem Cell retroVirus LTR promoter for mammalian gene knockdown
44248	pgRNA-humanized	Expression of customizable guide RNA (gRNA) under cotnrol of murine U6 promoter, also contains a CMV-puro-t2A-mCherry expression cassette for mammalian gene knockdown
44249	pdCas9-bacteria	aTc-inducible expression of a catalytically inactive bacterial Cas9 (S. pyogenes) for bacterial gene knockdown
44250	pwtCas9-bacteria	aTc-inducible expression of wild-type Cas9 (S .pyogenes) for bacterial gene knockdown
44251	pgRNA-bacteria	Expression of customizable guide RNA (gRNA) for bacterial gene knockdown