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. CTD2 researchers at the University of California, San Francisco recently addressed these shortcomings in a publication in Cell. They modified the CRISPR system to dynamically regulate gene repression (CRISPRi) and also 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 factors 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.