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Researchers use Modified CRISPR Systems to Modulate Gene Expression on a Genomic Scale

April 13, 2015, by Thomas Calder, Ph.D.

Crystal structure of Cas9 in complex with guide RNA and target DNA. Image adapted from work by Hiroshi Nishimasu, F. Ann Ran, Patrick D. Hsu, Silvana Konermann, Soraya I. Shehata, Naoshi Dohmae, Ryuichiro Ishitani, Feng Zhang, and Osamu Nureki, via Wikimedia Commons.

The genetic engineering system, clustered regularly interspaced short palindromic repeats (CRISPR), has conventionally been used to inactivate genes by making targeted double stranded cuts in DNA. While CRISPR is a useful tool, it can only be used to create loss-of-function modifications and often causes off-target effects due to the disruptive mechanism by which it works. 

Cancer Target Discovery and Development Network (CTD2) researchers at the University of California, San Francisco addressed these shortcomings in a publication in Cell. They modified the CRISPR system to dynamically regulate gene repression (CRISPRi) and established a novel system that results in gene activation (CRISPRa). 

These systems have a catalytically inactive CRISPR-associated protein 9 (Cas9) fused to either the gene expression inhibitor, Krüppel associated box (KRAB), or the gene expression activator, herpes simplex virus protein tetramer (VP64). By using Cas9 as a recruitment factor instead of a DNA nuclease, these systems reduce off-target effects that are normally associated with active Cas9.

The researchers found CRISPRi/a can be targeted to specific transcriptional start sites (TSS) with the addition of gene-specific small-guide RNAs (sgRNAs). They identified the optimal TSS target region for these sgRNAs by testing the ability of the CRISPRi/a systems to modulate gene expression in a Ricin challenge experiment, a commonly used validation test. With this knowledge, they designed and mass-produced sgRNAs to target the TSS of every protein-encoding gene in the genome. Analysis of CRISPRi/a on a genomic-scale established that these systems exhibit few off-target effects with low cellular toxicity, suggesting the CRISPRi/a systems may be useful tools for identifying gene functions in a high throughput manner.

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