Analogous to CRISPR/Cas9 knockout screening, CRISPR inhibition (CRISPRi) and CRISPR activation (CRISPRa) utilize RNA-guided targeting of Cas9 to genes of interest. However, these technologies are based on a catalytically dead mutant Cas9 (dCas9) and therefore cannot cut DNA. In CRISPRi, dCas9 is fused to a transcriptional repressor domain such as KRAB from ZNF10 or ZIM3. By targeting the dCas9-KRAB fusion protein to the promoter of a gene of interest, it is possible to suppress this gene’s expression. In contrast, CRISPRa utilizes dCas9 fusion proteins that promote transcription. While there are several distinct approaches, most involve use of the transcriptional activation domain VP64 . Moreover, in addition to direct protein fusion, transcriptional activators such as p65-HSF may be recruited to the gene of interest by adding binding sites for these transcriptional activators in the 3’ tracer sequence of the gRNA.

The benefits of using CRISPRi compared to CRISPR knockout include (i) absence of Cas9-mediated DNA damage that could trigger responses that confound the results of the experiment or screen, (ii) an incomplete suppression of gene expression, which could yield new insights with regard to essential genes, and (iii) temporal control over gene expression when using inducible dCas9-KRAB systems. The obvious advantage of CRISPRa is that it provides a new tool for studying the effects of increased gene expression, which previously required expression of cDNAs or ORFs.