What is CRISPRa vs. CRISPRi?

CRISPR-Cas9 for gene overexpression and down-regulation

The S. pyogenes CRISPR-Cas9 system is commonly used for gene knockout experiments. It consists of a Cas9 nuclease, responsible for creating a DNA double-strand break (DSB), and guide RNA, responsible for targeting the nuclease to a specific region in the genome. The cells’ endogenous DNA repair mechanisms imperfectly repair the DSB, leading to gene knockout.

The CRISPR-Cas9 gene knockout system has also been adapted into gene modulation technologies collectively known as CRISPR modulation (CRISPRmod), which includes CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). These technologies utilize nuclease-deactivated Cas9 (dCas9, Figure 1A) that binds to the target genomic region with the same efficiency as Cas9 but cannot generate a DSB and instead results in RNA-directed transcriptional control of the target region. CRISPRi^1,2utilizes dCas9 with or without fused repressor domains along with a standard S. pyogenes guide RNA to target the promoter regions for transcriptional repression, or knockdown, of a gene. In contrast, CRISPRa^3-8 employs dCas9 fused to transcriptional activation domains, which can be directed to promoter regions by either standard S. pyogenes guide RNA or special guide RNAs that recruit additional effectors for transcriptional activation and increased expression of the target gene (Figure 1B).

crispri crispra mutations transcription — Figure 1. A. CRISPRi and CRISPRa use deactivated or “dead” Cas9 (dCas9), a nuclease-deficient mutant variant of the Cas9 protein. The schematic of dCas9 shows two point mutations (D10A, H840A) that inactivate the DNA cleavage activity. B. CRISPRi and CRISPRa technologies essentially generate artificial transcription factors by attaching an effector domain to dCas9 to silence or activate transcription.

Figure 1. A. CRISPRi and CRISPRa use deactivated or “dead” Cas9 (dCas9), a nuclease-deficient mutant variant of the Cas9 protein. The schematic of dCas9 shows two point mutations (D10A, H840A) that inactivate the DNA cleavage activity. B. CRISPRi and CRISPRa technologies essentially generate artificial transcription factors by attaching an effector domain to dCas9 to silence or activate transcription.

Challenges in guide RNA design for transcription start sites

CRISPRi or CRISPRa requires guide RNA designs in proximity to the gene’s promoter region or the transcriptional start site (TSS) to result in silencing or activation, respectively^4,6. Designing functional guide RNAs can be challenging, as TSS are not always well annotated, they may be inaccessible due to the presence of other protein factors, and cryptic or alternative gene promoters may be utilized. Fortunately, genome-wide studies that have systematically manipulated gene expression using pooled single guide RNA (sgRNA) screens have enabled the development of design algorithms with improved guide RNA functionality for both CRISPRi and CRISPRa⁹.

Ordering CRISPRi/a reagents

Horizon offers CRISPRi guide RNA and CRISPRa guide RNA in both synthetic and lentiviral expressed formats – providing flexibility in experimental workflow design.

The CRISPRi system uses a novel dCas9-SALL1-SDS3 repressor we developed in-house, which is the first repressor on the market to provide robust and consistent gene repression using either synthetic guide RNAs or vector-based guide RNAs. dCas9-SALL1-SDS3 shows greater repression in the majority of genes tested when compared to other repressor systems currently available on the market.

Our CRISPRa products use the dCas9-VPR activation system⁷, and both CRISPRa/i utilize predesigned guide RNAs based on a published CRISPR algorithm⁹. Guide RNAs can be ordered on the Horizon website by simply searching for the gene of interest and choosing the preferred format (e.g. lentiviral expressed guide RNA or synthetic guide RNA). Alternatively, custom self-designed S. pyogenes guide RNA for CRISPRi and CRISPRa systems can be ordered through the CRISPR Design Tool using rules defined in the *literature^{4,8 9}.

*The Horizon sgRNA vector is compatible with CRISPRi and CRISPRa systems that use canonical guide RNA^2,3,4,7, but not the systems that use modified effector-binding scaffold sgRNAs^6,7.

When should I use CRISPRi and CRISPRa?

CRISPRmod technology allows for activation and repression of gene transcription in the endogenous context, and is applicable to both coding and noncoding genes. It can also enable whole genome activation and interference screening in a pooled format. To achieve activation or repression using this method, delivery or expression of both the dCas9 protein and a guide RNA is required.
CRISPRi silences genes at the transcriptional level, and in general may have fewer sequence-specific off-target effects than RNAi. One of the main benefits of CRISPRi is the ability to pool guide RNAs for enhanced repression of an individual gene, while multiplexing guide RNAs from different genes allows simultaneous repression of multiple genes to examine complex cellular pathways and interaction networks. CRISPRi may be complementary to CRISPR-Cas9 knockout and RNAi-based approaches, meaning it can be used for orthogonal validation of these technologies. CRISPRi is also suitable for studying long noncoding RNAs^10,11.
CRISPRa activates genes at the transcriptional level. For the first time, this technology allows for overexpression of genes in their endogenous context and is applicable to both coding and noncoding genes. While other technologies such as plasmid and lentiviral-based ORFs may still be a good option for robust, exogenous overexpression of a single gene expression construct, CRISPRa technology enables endogenous overexpression of large transcripts for which ORF overexpression is not possible.

References

L. S. Qi et al., Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 152, 1173–1183 (2013).
L. A. Gilbert et al., CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell. 154, 442–451 (2013).
A. W. Cheng et al., Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).
L. A. Gilbert et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 159, 647–661 (2014).
M. E. Tanenbaum, L. A. Gilbert, L. S. Qi, J. S. Weissman, R. D. Vale, A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 159, 635–646 (2014).
S. Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 517, 583–588 (2015).
A. Chavez et al., Highly efficient Cas9-mediated transcriptional programming. Nat. Methods. 12, 326–328 (2015).
J. G. Zalatan et al., Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell. 160, 339–350 (2015).
M. A. Horlbeck et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife. 5, e19760 (2016).
S. J. Liu et al., CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science. 355 (2017), doi:10.1126/science.aah7111.
A. Goyal et al., Challenges of CRISPR/Cas9 applications for long non-coding RNA genes. Nucleic Acids Res. (2016), doi:10.1093/nar/gkw883.

Authors: Jennifer Abarca is a Scientific Support Scientist, Zaklina Strezoska is a Senior Scientist II, and Annaleen Vermeulen is a Senior Scientist II at Horizon Discovery.