The recent repurposing of the CRISPR-Cas9 bacterial immune system has brought about a revolution in genome engineering and functional genomics—by targeting the Cas9 endonuclease with a short guide RNA (sgRNA), a double-strand break is introduced at the desired loci resulting in robust and efficient gene knockout (CRISPR-ko). The simplicity and efficiency of the CRISPR-Cas9 system has seen the technology rapidly adopted by laboratories and scientists, and within a year of the first demonstration of genome editing in mammalian cells, CRISPR-Cas9 was successfully utilized as a functional genomic screening tool to perform loss-of-function studies on a genome-wide scale.1,2 But since then, the CRISPR-Cas9 toolkit has been expanded—using a modified version of Cas9, transcriptional silencing and upregulation of gene expression is now possible with CRISPRi and CRISPRa. This article will discuss the benefits of CRISPRi and CRISPRa and what this additional CRISPR functionality brings to functional genomic screening.

Expanding the CRISPR toolbox

Functional genomic screening utilizing CRISPR-Cas9 gene-editing technology now allows whole-genome screening to be administered in a pooled format within the technical capabilities of most laboratories. Drawing on methods developed with RNAi, libraries of sgRNA that knock out every gene in the genome are delivered via lentiviral vector that stably integrate into the genome. This effectively provides each cell with a “barcode”, enabling identification of those sgRNA enriched or depleted after the screen via PCR and next-generation sequencing. Functional genomic screening with CRISPR-ko has enabled elucidation of biochemical pathways and drug target identification, allowing scientists to address key questions about cellular pathways and regulation mechanisms.

But further development of the CRISPR-Cas9 technology now means that other genetic perturbations are now possible—as well as gene knockout, researchers can now produce transcriptional silencing and activation of gene expression, while capitalizing on the high specificity and precision of the CRISPR-Cas9 system. The use of a catalytically inactive version of Cas9 (dead Cas9, or dCas9) targeted to prokaryotic promoter regions was found to result in inhibition of gene expression due to steric hindrance of transcriptional machinery—in mammalian cells, fusion of repressor domains such as KRAB to dCas9 is required for transcriptional silencing.3,4 This process is termed CRISPR interference, or CRISPRi, and as there is no change to the DNA, CRISPRi allows targeted but reversible knockdown rather than knockout.

Using this approach, dCas9 fusions can also be used to activate gene expression, with CRISPR activation, or CRISPRa. dCas9 fused to transcriptional activators such as VP64 and p65 can be targeted to promoter and enhancer regions, resulting in upregulation of gene expression. To enhance gene activation in mammalian cells so that a single sgRNA provides effective activation of gene expression, dCas9 fusions that recruit multiple activator domains are required. Several systems have been developed, including direct dCas9 fusions (e.g. VPR, a tripartite fusion of VP64, p65, and Rta5), utilization of a protein scaffold (e.g. SunTag system6), and those incorporating an RNA scaffold (e.g. Synergistic Activation Mediator complex (SAM207)).

Functional genomic screening with CRISPRi and CRISPRa

Shortly after the publication of the first CRISPR-ko screens, and perhaps somewhat inevitably, CRISPRi and CRISPRa techniques were scaled up to the genome level.8 This proof-of-principle work by Gilbert et al., demonstrated that the CRISPRi/a technology could be used to elicit genome-wide transcriptional modulation that is inducible and reversible, identifying genes that control cellular responses to the chimeric choleria/diptheria fusion toxin (CTx-DTA). Initial proliferation screens using CRISPRi compared favorably to RNAi and yielded complementary results to CRISPR-ko, returning “gold standard” essential genes, such as DNA replication genes and ribosomal subunits.9

However, constitutive binding of dCas9 is affected by nucleosomes, and so dCas9 fusions may not achieve full functionality if blocked by histone-DNA binding. Using screening data and transcriptional start site analysis to optimize sgRNA positioning for nucleosome free regions, Horlbeck et al., generated a highly optimized sgRNA design platform algorithm to identify the best sgRNA for CRISPRi and CRISPRa applications.10 This development considerably improves efficiency and specificity of CRISPRi, vastly improving performance over RNAi to match that of CRISPR-ko.

CRISPRi and CRISPRa remain relatively young technologies and have yet to see the wide adoption of CRISPR-ko, or the established technology of RNAi. However, some early publications are demonstrating the utility of these approaches:

Long non-coding RNAs: CRISPRi has shown itself suited to studying long non-coding RNAs, and when combined with droplet-based, single-cell RNA sequencing, has been used to analyze the molecular pathways that guide cellular differentiation.11 Additionally, because CRISPRa is not contingent on targeting exons of protein coding genes, scientists have also developed CRISPRa libraries to target both coding and non-coding regions at the same time to identify functional coding/lncRNA resistance gene pairs.12

Up, down and out with CRISPR-Cas9

  • The CRISPR-Cas9 system has proven to be an invaluable and versatile tool for researchers, coupling high specificity and precision, with ease of use.
  • Unbiased and systematic interrogation of genomes with CRISPR-Cas9 knockout in a pooled format now only requires basic molecular techniques and access to next-generation sequencing and analysis.
  • A modified version of Cas9 has added to the functionality of the CRISPR-Cas9 system—modulation of gene expression with CRISPRi and CRISPRa can now be used to study changes to transcription on a genome-wide scale.
  • The versatility of the CRISPR system means that CRISPR-ko, CRISPRi, and CRISPRa can be used individually or in combination for high-throughput genetic screening to determine gene function, link genotype with phenotype, and further our understanding of the molecular basis of biological pathways and disease.

Drug resistance: CRISPRa screening of BRAF V600E mutant cells was used to study the mechanisms of resistance to the BRAF inhibitor PLX-4720. The screen identified previously known resistance mechanisms, such as EGFR and ERK pathway activation, but also revealed novel resistance mechanisms involving G protein-coupled receptors.7

Elucidation of cellular pathways: CRISPRa screening has also been used to identify 50 novel reprogramming factors in mouse epiblast stem cells to induce pluripotency.13

Precise control of gene expression

Although CRISPR-ko is a powerful tool to use in a pooled genetic screen, gene knockout does have its limitations. CRISPR-ko produces a true null phenotype, useful when screening weaker phenotypes that could be masked by residual low-level protein expression, but not suited to studying essential genes or pharmacological inhibition by a drug. Drug suppression of gene products is rarely absolute, and so partial knockdown of gene expression, rather than total knockout, would be better placed to mimic the action of a drug and to understand the cellular intricacies of gene expression on target sensitivity. CRISPR-ko is also limited in its ability to probe the role of gene activation mutations in biology and disease as it only addresses loss-of-function effects. As genes are frequently activated during cancer (i.e. KRAS, BRAF), CRISPRa has enormous potential for studying the molecular pathology of the disease, as well as drug-resistance mechanisms that are caused by gain of function events.

Scientist now also have the option to combine a CRISPR-ko screen with CRISPRi or CRISPRa. These can be performed simultaneously therefore economizing use of time, plus because the approaches are orthologous, this can increase confidence in hits identified in both screens. Additionally, it is likely that the different screens will also identify different hits, expanding the pool of potential targets that scientists can explore as part of the target-identification efforts.

References

1. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343,84–87 (2014).

2. Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic Screens in Human Cells Using the CRISPR-Cas9 System. Science (New York, N.Y.) 343, 80–84 (2014).

3. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

4. Gilbert Luke A et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

5. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nature Methods12, 326–328 (2015).

6. Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159,635–46 (2014).

7. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–8 (2015).

8. Gilbert, L. A. et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell 159, 647–661 (2014).

9. Hart, T., Brown, K. R., Sircoulomb, F., Rottapel, R. & Moffat, J. Measuring error rates in genomic perturbation screens: gold standards for human functional genomics. Molecular Systems Biology10, 733–733 (2014).

10. Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife 5,(2016).

11. Genga, R. M. J. et al. Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves Molecular Drivers of Early Human Endoderm Development. Cell Reports 27, 708-718.e10 (2019).

12. Bester, A. C. et al. An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance. Cell 173, 649-664.e20 (2018).

13. Yang, J. et al. Genome-Scale CRISPRa Screen Identifies Novel Factors for Cellular Reprogramming. Stem Cell Reports 12, 757–771 (2019).