CRISPR-based platform for RNA-guided DNA targeting

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A naturally observed phenomenon in prokaryotic organisms is the CRISPR/Cas adaptive immune system for site-specific DNA cleavage.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants.

The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting (allowing for robust and precise targeting of genes for expression or repression).

Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes with the site of delivery determined solely by a coexpressed short guide (sg)RNA. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific and has been shown to enable robust gene repression and activation in human cells (1, 2).

Both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). A recent study provides evidence about how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA (3).

(1) CRISPR RNA-guided activation of endogenous human genes. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. Nat Methods. 2013 Oct;10(10):977-9. PMID: 23892898

(2) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Gilbert et al. Cell. 2013 Jul 18;154(2):442-51.  PMID: 23849981

(3) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. Nature. 2014 Mar 6;507(7490):62-7. PMID: 24476820

Posted by Yannis Trakadis, MD

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