CRISPRa and CRISPRi: tracing the origins and exploring the future.

As we reach the mid-2020s, the CRISPR-Cas9 system continues to revolutionize functional genomics and genetic screening. In recent years, sophisticated tools based on dCas9, particularly CRISPRa (activation) and CRISPRi (interference), have emerged, transforming our ability to study gene function. These cutting-edge techniques allow researchers to rapidly modulate gene expression, enabling more nuanced investigations into drug targets and complex biological processes.

The ongoing evolution of CRISPR technology is opening new avenues for therapeutic development and providing deeper insights into genetic regulation. As we look to the future, these advancements promise to accelerate discoveries in personalized medicine and broaden our understanding of the human genome.

The origins

January 2013 marked a breakthrough in genome engineering. Four laboratories simultaneously engineered the bacterial and archaeal CRISPR-Cas9 system to induce precise cleavage at mammalian genomic loci^1-4. Within a year, two consecutive papers documented the application of CRISPR-Cas9 knockout technology to forward genetic screening, cornerstones in life sciences that demonstrated the potential of this new technology. These studies not only proved the concept of this technology but also represented a remarkable leap in the capabilities of functional genomics^5-6. Since then, hundreds of studies have built upon these discoveries, including some published by our scientists^7-8. These investigations have introduced screening platforms with increasing precision and performance.

From gene editing to gene regulation

Knockout CRISPR was only the beginning. Inspired by modified ZnF nucleases and TALEs, researchers engineered a nuclease-dead version of Cas9 (dCas9)⁹. Rather than mutating specific genomic loci, dCas9 allowed disruption of gene transcription simply by binding to proximal sequences at the gene's promoter region. This system, known as CRISPR interference (CRISPRi), efficiently blocked transcript initiation in E. coli and mammalian cells^10-14. Researchers achieved enhanced transcriptional silencing by covalently linking the KRAB transcriptional repressor to dCas9^1,4.

Using the dCas9 approach, it also became possible to create a transcriptional activation tool, coined CRISPR activation (CRISPRa), by fusing dCas9 to the VP64 and p65 activation domains^14,16-18. Effective gene activation with CRISPRa was accomplished by several approaches: the SunTag array, which used multiple VP64s recruited onto a peptide array¹⁹; VPR, a synergistic tripartite activation method using a fusion of VP64, p65, and Rta²⁰; and the Synergistic Activation Mediator complex (SAM²¹), which used a dCas9-VP64 fusion and recruitment of p65 and HSF1 via RNA binding protein components. These adapted CRISPR tools provided incredible new opportunities to study gain-of-function mutations in genome-wide screens^15-21.

Enhancing dCas9

For dCas9 to achieve full functionality, it had to exert its activating or inhibitory effect on gene output through constitutive binding. Intriguingly, Hinz et al., Horlbeck et al., and Isaac et al. discovered that dCas9 activity was influenced by nucleosome occupancy and that histone-DNA binding effectively blocked guide RNA access. By coupling data from multiple CRISPRi and CRISPRa screens to transcription start-site analysis (FANTOM), Horlbeck et al. could generate highly optimized versions of their guide RNA design platforms. This advance in technology substantially increased the efficiency of these tools. It brought hit identification by pooled CRISPRi screening to a performance level that could exceed CRISPR knockout.

Choosing the best dCas9 strategy

With all these exciting new tools available, picking the right one for the right job was crucial for research success. In principle, knocking out genes rather than repressing their expression provided the most significant potential window for discovery; however, the recent adaptations to CRISPRi guide RNA design leveled the playing field. CRISPR knockout wasn't ideally suited to studying hypomorphic phenotypes, including essential genes, and complete gene knockout might not have been the best model for druggability. But knockout technology's high precision and penetrance provided unprecedented clarity and outstanding quality datasets. One chink in the armor of CRISPR knockout was its application to amplified loci, where multiple cuts could cause an off-target DNA damage response.

Additionally, since dCas9 did not alter the sequence of genomic DNA, its activity could be made inducible and reversible, which was not the case for the Cas9 nuclease. CRISPRi and CRISPRa approaches were also more suitable for studying the differential expression of long noncoding RNAs. These genes had proven difficult to target effectively with the CRISPR knockout platform. Unique to CRISPRa was the exciting prospect of studying gain-of-function phenotypes, opening up myriad new paths to discovery.

With the advancements in CRISPRi technology, Revvity researchers were able to generate stable cell lines that repressed their genes of interest. The CRISPRmod interference system emerged as a unique adaptation of the classical CRISPR-Cas9 gene editing system. This system utilized a catalytically deactivated Cas9 (dCas9) fused to repressor domains such as SALL1 and SDS3. It effectively reduces transcription when paired with a well-designed guide RNA targeting a gene near its promoter region or transcriptional start site (TSS).

Similarly, the CRISPR activation (CRISPRa) system represented a unique adaptation of the classical CRISPR-Cas9 gene editing system. It utilized a catalytically deactivated or dead S. pyogenes Cas9 (dCas9) fused to one or more transcriptional activators. It promoted transcriptional activation when paired with a well-designed guide RNA targeting a gene near a promoter region or TSS.

With the greatly improved CRISPRi and CRISPRa technology enabling researchers to explore novel areas of biology that are generally out of reach with knockout technology, exciting times lay ahead.