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-stranded break (DSB), and guide RNA (gRNA) that is responsible for targeting the nuclease to a specific region in the genome. The cells’ endogenous repair mechanisms can imperfectly repair the DSB and lead to a gene knockout. The CRISPR-Cas9 system has also been adapted to generate technologies called CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation). These utilize nuclease-deactivated Cas9 (dCas9) that cannot generate a DSB (Figure 1A), but instead target genomic regions resulting in RNA-directed transcriptional control. CRISPRi1,2 utilizes dCas9 with or without a KRAB effector domain that complexes with gRNA to target promoter regions for transcriptional repression, or knockdown, of the gene. CRISPRa3-8 employs dCas9 fused to different transcriptional activation domains, which can be directed to promoter regions by either standard S. pyogenes gRNA or special gRNAs that recruit additional transcriptional activation domains to upregulate expression of the target gene (Figure 1B).
Challenges in guide RNA design for transcription start sites
CRISPRi or CRISPRa requires sgRNA designs in proximity to the gene’s promoter region or the transcriptional start site to result in silencing or activation, respectively.4,6Designing functional gRNAs can be challenging, as transcriptional start sites are not always well annotated or may be inaccessible due to presence of other protein factors, and cryptic or alternative promoters may be utilized. However, systematic manipulation of gene expression at genome scale utilizing pooled sgRNA screening has enabled development of design algorithms with improved gRNA functionality for CRISPRi and CRISPRa.9
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.
Ordering single guide RNAs for CRISPRi or CRISPRa
Most CRISPRi and CRISPRa publications to date have used expressed single guide RNA (sgRNA) (e.g., plasmid or lentivirus), but custom or predesigned synthetic guide RNA oligos provide a convenient and fast cloning-free alternative.
Dharmacon CRISPRa products utilize the dCas9-VPR activation system7, and guide RNAs based on a published CRISPR algorithm9. Predesigned guide RNAs for human and mouse protein coding genes can be ordered on the Dharmacon website by simply searching for the gene of interest and choosing your preferred format (expressed sgRNA or synthetic crRNA). 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 Dharmacon sgRNA vector is compatible with CRISPRi and CRISPRa systems that use canonical guide RNA2,3,4,7, but not the systems that use modified effector-binding scaffold sgRNAs.6,7
When should I use CRISPRi and CRISPRa?
- CRISPRi silences genes at the transcriptional level. One of the potential benefits is this technology may have fewer sequence-specific off-target effects than RNAi, and is applicable to both coding and noncoding genes. The drawback of this technology is that silencing requires delivery of both the dCas9-KRAB protein and an RNA. This technology may be complimentary to RNAi and CRISPR-Cas9 knockout approaches 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. Other technologies such as plasmid and lentiviral-based ORFs may still be a good option for robust, exogenous overexpression. CRISPRa technology, however, enables overexpression of large transcripts for which ORF overexpression is not possible. It also enables whole genome activation screening in pooled format. The drawback of this technology is that activation requires delivery or expression of both dCas9-activator and a guide RNA, as opposed to a single gene expression construct.
- 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 Technical Support Scientist, Zaklina Strezoska is a Senior Scientist II, and Annaleen Vermeulen is a Senior Scientist II at Dharmacon, Inc.
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