CRISPR interference

CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells.

[1] It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna.

Based on the bacterial genetic immune system - CRISPR (clustered regularly interspaced short palindromic repeats) pathway,[3] the technique provides a complementary approach to RNA interference.

Many bacteria and most archaea have an adaptive immune system which incorporates CRISPR RNA (crRNA) and CRISPR-associated (cas) genes.

The CRISPR interference (CRISPRi) technique was first reported by Lei S. Qi and researchers at the University of California at San Francisco in early 2013.

Secondary variables must also 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 ensuring that no restriction sites are present in the modified sgRNA, as this may pose a problem in downstream cloning steps.

For example, the well-studied Krüppel associated box (KRAB) domain can be fused to dCas9 to repress transcription of the target gene up to 99% in human cells.

[17] Along with other improvements mentioned, factors such as the distance from the transcription start and the local chromatin state may be critical parameters in determining activation/repression efficiency.

[2] A significant portion of the genome (both reporter and endogenous genes) in eukaryotes has been shown to be targetable using lentiviral constructs to express dCas9 and sgRNAs, with comparable efficiency to existing techniques such as RNAi and TALE proteins.

For bacteria, gene knockdown by CRISPRi has been fully implemented and characterized (off-target analysis, leaky repression) for both Gram-negative E. coli [4][6] and Gram-positive B.

Activation of Yamanaka factors by CRISPRa has been used to induce pluripotency in human and mouse cells providing an alternative method to iPS technology.