Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System.

Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, Park J, Blackburn EH, Weissman JS, Qi LS, Huang B
Cell. 2013 Dec 19;155(7):1479-91. doi: 10.1016/j.cell.2013.12.001. 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 mammalian gene knockdown

The first plasmid (pdCas9_humanized) contains a human codon optimized dCas9 gene under the control of Murine Stem Cell retroVirus LTR promoter. The second plasmid (pgRNA_humanized) contains a murine U6 promoter controlled sgRNA cassette, wherein the GN19 can be custom designed to target sequences in the genome. The pgRNA_humanized also contains a CMV-puro-t2A-mCherry expression cassette, useful for selection or fluorescent gating of transiently transfected cells. Co-expression of both plasmids in HEK293 cells could cause up to 2~3-fold repress on targeted fluorescent genes.

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

Plasmids from Article

ID	Plasmid	Purpose
51023	pSLQ1658-dCas9-EGFP	Template for NLS-dCas9-NLS-EGFP fusion protein for CRISPR imaging (the recipient vector can be TetON 3G promoter system)
51024	pSLQ1651-sgTelomere(F+E)	Lentiviral vector that contains an optimized S. pyogenes sgRNA targeting human telomeres
51025	pSLQ1661-sgMUC4-E3(F+E)	Lentiviral vector that contains an optimized S. pyogenes sgRNA targeting the repetitive sequence of human MUC4 exon 3