CRISPR Cas9 - Gene Regulation with dCas9

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Gene Regulation with dCas9

Gene Regulation with dCas9

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Video Summary

In this knowledge base, we will introduce you to yet another way scientists have modified the CRISPR Cas9 system - this time to use it as a platform for the modular attachment of genetic and epigenetic regulators.

dCas9 as a Tool for Transcriptional Modulation

CRISPR Cas9 - Applications for Gene Regulation

Scientists the world over have marveled over CRISPR’s remarkable simplicity and versatility as a gene editor—but “killing” it’s catalytic activity might be an even more brilliant idea! In 2013, Qi et al. mutated the nuclease domains of Cas9 from S. pyogenes (making an H840A mutation in the HNH domain and a D10A mutation in the RuvC domain) to create a nuclease deficient “dCas9” (1) (also called dCas9 null mutant). Although this “blunted” and “dead” version of Cas9 is no longer able to cleave DNA, it can still target and bind DNA with the same precision when guided by sgRNA. But instead of irreversibly altering the genome, binding of dCas9 interferes with the transcription of the target site—resulting in the reversible silencing of the gene.

The use of dCas9 itself as a method of transcriptional interference was only the beginning. Soon after, researchers also began attaching effectors (such as repressor proteins and activator domains) to harness dCas9’s targeting abilities for reversible gene activation, epigenomic editing, and much more. Whether it is a promoter region, regulatory region, or coding region, scientists can use the CRISPR dCas9 system as a modular scaffold for easy effector attachment, enabling the control of any gene without introducing irreversible DNA-damaging mutations.

CRISPR Cas9 - Gene Repression and Activation

Figure 1 – dCas9 as a modular system for attachment of transcriptional regulators. dCas9 can easily be fused to effectors (either transcription activators or repressors) for targeted gene regulation. Adapted from Figure 1a of Gilbert et al. (2013).

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dCas9 Mediated Gene Activation and Repression

Scientists the world over have marveled over CRISPR’s remarkable simplicity and versatility as a gene editor—but “killing” it’s catalytic activity might be an even more brilliant idea! In 2013, Qi et al. mutated the nuclease domains of Cas9 from S. pyogenes (making an H840A mutation in the HNH domain and a D10A mutation in the RuvC domain) to create a nuclease deficient “dCas9”(1) (also called dCas9 null mutant). Although this “blunted” and “dead” version of Cas9 is no longer able to cleave DNA, it can still target and bind DNA with the same precision when guided by sgRNA. But instead of irreversibly altering the genome, binding of dCas9 interferes with the transcription of the target site—resulting in the reversible silencing of the gene.

a. dCas9-SAM—Repurposing the CRISPR Cas9 system for gene activation

In order to endow dCas9 with gene activation abilities, dCas9 was first fused with classic transcriptional activators such as VP64 (a synthetic tetramer of the Herpes Simplex Viral Protein 16) or p65 (a transcription factor involved in many cellular processes) (2). Although these single gene regulator systems demonstrated gene activation across various eukaryotic cells, only moderate activation was achieved (2-5 fold) (2).

In an effort to enhance activation power, the synergistic activation mediator (SAM) system was developed (3). This system builds upon the basic dCas9-VP64 structure, but includes an sgRNA modified to recruit additional transcriptional activators for a synergistic activation effect. This modified sgRNA incorporates two RNA hairpin aptamers that bind to dimers of the bacteriophage MS2 coat proteins (2). Fusion of the MS2 proteins to additional activators such as p65 and the human heat shock factor 1 (HSF1) (2) results in the recruitment of 13 activation molecules per dCas9 molecule (Figure 2a). This new dCas9-SAM system can reliably amplify gene expression from 10 to multiple thousand-fold, depending on baseline expression (2) (Figure 2b).

Building off of this idea, Church et. al designed a system in which the tripartite activation system is made of a fusion of VP64, p65, and RTA—a system referred to as the dCas9-VPR system (20). dCas9-VPR does not require a modified sgRNA for effective activation, simplifying the design process significantly. Although gene activation was comparable between the dCas9-SAM and VPR system in general, the VPR system demonstrated superior activation in U-2 OS and MCF7 cells (22). When combined with an sgRNA library, this system is also able to support large scale, genome-wide gain-of-function screens, making it a powerful tool for studying biological processes and pathways (3).

The dCas9-SAM system is an elegantly simple method for scientists to selectively upregulate gene expression at a specific target within its native chromosomal context, making it an important tool for basic research advancement. Because of its robust activation ability, this system would be an unparalleled transformative tool in the development of therapeutic interventions, genetic screening, and transcriptional manipulation of endogenous and synthetic genetic circuits (4) across a variety of cell types (22). Researchers are already making use of the dCas9-SAM system in the activation of HIV-1 transcription to induce apoptosis for cell destruction (5) as well as in the induction of dormant HIV-1 proviral DNA for its complete elimination (6).

Figure 2 – The dCas9-SAM transcriptional activation system. a) The dCas9-SAM system is made up of a dCas9 fused to the transcriptional activator, VP64. The accompanying sgRNA can also be modified to contain two RNA aptamers for binding with MS2 coat proteins that are also fused to one p65 and one HSF1 transcription activator. Adapted from Figure 1f of La Russa et al. (2015). b) A comparison of the activation efficiency of dCas9-VP64 by itself (yellow) as well as with the modified sgRNA system (green) in the activation of four different genes: HBG1, IL-1B, IL1R2, and ZFP42. Relative expression levels were quantified using qPCR, with fold changes determined by comparing with GFP-transfected cells. Adapted from Figure 1b of Konermann et al. (2015). c) The dCas9-VPR system is composed of an ordered fusion of transcriptional activators VP64, p16, and RTA. This system does not require a specially modified sgRNA to achieve the same activation capability as the dCas9-SAM system. Adapted from Figure 1a of Chavez et al. (2016). d) A comparison of the activation performance of dCas9-VP64, dCas9-VPR, and dCas9-SAM of the RHOXF2 gene.

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b. Modifying the CRISPR Cas9 system for transcriptional repression

dCas9 alone is capable of targeted inhibition of gene transcription as its binding to the target site sterically interferes with the binding and function of transcriptional machinery, a method called CRISPRi (or CRISPR Interference). This simple CRISPRi system can effect up to 1,000-fold repression, efficiently knocking down gene expression in cells (4). Although this system performs fairly well in bacteria, yeast, and other prokaryotic cells, it is less effective at repressing gene expression in mammalian cells (7).

This challenge led to the development of the dCas9-KRAB system (7)—a system where dCas9 is fused with KRAB (Krüppel-associated box), the transcriptional repressor domain of Kox1 (Figure 3a). This enhanced CRISPRi system relies on KRAB’s ability to recruit a diverse array of histone modifiers that reversibly suppresses gene expression through the formation of heterochromatin. Using this system, a 60-80% reduction in the expression of highly specific endogenous eukaryotic genes was achieved during transient transfection (Figure 3b) (7). Furthermore, stably integrated dCas9-KRAB in HeLa cells caused a robust 5-10 fold repression of endogenous genes and promoter regions (7), with a 100-fold effect observed when the target site was 50-100 bp downstream of the transcription start site (1). dCas9-KRAB also showed no effect on cell growth, making it a nontoxic method for gene silencing (8).

Unlike other classical gene silencing approaches such as RNAi (a method that knocks down gene expression via degradation of transcribed mRNA in the cytoplasm) (9), the dCas9-KRAB system offers reversible inhibition at the DNA level. This enables highly specific gene repression as well as silencing of non-coding RNAs, microRNAs, antisense transcripts, and nuclear localized RNAs (7). Future work on expanding the dCas9-KRAB system for addition of other repressor partners may serve to amplify the silencing power even further.

Figure 3 – The dCas9-KRAB transcriptional repression system. a) dCas9 can be fused to KRAB, a transcriptional repressor. Adapted from Shalem et al. (2015). b) A comparison of relative CD71 expression levels in a dCas9 (blue) vs. a dCas9-KRAB (red) system targeted to CD71 using three different sgRNAs. Relative expression levels were determined from flow cytometry for CD71 protein expression. Adapted from Figure 3c of Gilbert et al. (2013).

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dCas9 Mediated Epigenetic Editing

With the advent of genome engineering technologies, we have been able to gain a better understanding of how genes give rise to select phenotypes. Overlaid on top of the genome is the epigenome, a second level of genomic regulation that involves modifications of both the nucleosome and the DNA base itself. Epigenetic regulation works by affecting the structure of a stretch of chromatin, either by compressing it into a compact and transcriptionally-active state (heterochromatin) or by oepning it up for expression. Years of efforts in functional genomics has enabled the mapping and characterization of millions of these epigenetic regulatory elements in different tissues and cell types, however, current methods for single-locus probing are laborious, expensive, and toxic to living cells.

dCas9 fusions have recently emerged as an unparalleled tool for the interrogation of the epigenome at individual loci (Table 1). In this method, dCas9 is fused to a growing range of different effector domains that can be easily switched out(21). This dCas9 epigenetics toolbox enables scientists to study and manipulate gene regulation without altering the underlying gene sequences. Systems such as these dCas9 fusions can help to further our understanding of the role of epigenetic changes in different molecular pathways and diseases and perhaps even correct such diseases.

Table 1 — The dCas9 Epigenetics Toolbox

	Construct	Function	Gene Expression
Histone Modifications	dCas9-p300	Acetylation	Activation
Histone Modifications	dCas9-LSD1	Demethylation	Repression
DNA Methylation	dCas9-TET1CD	Demethylation	Activation
DNA Methylation	dCas9-DNMT3A	Methylation	Repression

a. Manipulating histones with dCas9 systems

i. dCas9-p300—Epigenetic activation via a modified CRISPR Cas9 system

In order to gain a clearer understanding of the mechanisms behind gene regulation, tools that can modulate epigenetic tags with high specificity are needed. Technologies such as histone deacetylases or DNA methyltransferases fused to Zinc finger proteins or transcription activator-like effectors (TALEs) were created to modify the epigenome via targeted demethylation and deacetylation(10). However, histone acetylation is one of the most powerful gene expression enhancer systems. Hilton et al. developed the dCas9-p300 system in order to meet this need(11), enabling the direct modification of the chromatin state involved in a broad range of cellular pathways and processes.

In this system, dCas9 is fused to the catalytic core of the human E1A-associated protein p300, the key component of the domain that acetylates histones (Figure 4a)(11). This resulting system successfully induces gene expression when targeted to either coding or regulatory regions, demonstrating its effectiveness as a transactivator of downstream genes(11). In particular, activation was observed to be 50 (for the OCT4 and MYOD promoters) to 10,000 fold (for the IL1RN promoter, Figure 4b) when targeted to promoters or enhancers (10,11). Gene activation was highly specific as assessed by transcriptome profiling, indicating that single chromatin changes are enough for activation (or silencing). Because this system employs mammalian p300, it also has minimal immunogenicity potential, making it advantageous for in vivo applications.

dCas9-p300 is a simple and unique tool for mapping out the complex relationships between the epigenome, regulatory elements, and the target gene’s expression in functional genomics studies. By combining dCas9-p300 with inducible control, researchers will be able to activate genes in real time, enabling applications in genome-wide screens of regulatory element activity.

Figure 4 – The dCas9-p300 system for epigenetic activation. a) dCas9 fused to the core catalytic domain of p300 can acetylate target sites in the genome. Acetylation of the target gene synergizes with the action of transcription factors and RNA Polymerase II, resulting in transcriptional upregulation. Adapted from Figure 1b of Zentner et al. (2015). b) Comparison of relative expression levels of IL1RN when dCas9 by itself vs. dCas9 fused to the p300 catalytic core is targeted to the IL1RN promoter. Relative expression levels determined by qRT-PCR. Adapted from Figure 1c of Hilton et al. (2015).

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ii. dCas9-LSD1—Epigenetic repression via a modified CRISPR Cas9 system

dCas9-LSD1 is a complementary gene repressing system to the activating powers of dCas9-p300. In this system, dCas9 is fused to the Lysine-specific histone demethylase 1 (LSD1) (Figure 5a) (12). Using a mouse embryonic stem cell line that stably expressed dCas9-LSD1, Kearns et al. successfully demonstrated downstream gene repression when the system was targeted at the distal enhancer region of the Oct4 gene (12). However, when dCas9-LSD1 was targeted at the Oct4 promoter, no effect was observed. This makes dCas9-LSD1 a promising tool for studying the regulatory activity of enhancers. This is in contrast to other systems such as dCas9-KRAB, which are more global controllers of gene expression. dCas9-LSD1 was also able to silence the gene expression of the Tbx1 gene when targeted to its upstream enhancer (Figure 5b) (12) without disrupting the local genomic architecture. Ultimately, this silencing was shown to affect the cellular state of the embryonic stem cells (e.g. colony morphology and increase of differentiation-associated markers) (12).

Since many genomic regions associated with human diseases are found within enhancer regions, dCas9-LSD1’s ability to functionally annotate enhancer elements in a highly specific manner makes it invaluable in the quest towards defining enhancer-gene relationships. When used in combination with an enhancer-targeted sgRNA pool, this system can provide a high-throughput and systematic way to identify all enhancers related to a gene.

Figure 5 – The dCas9-p300 system for epigenetic activation. a) dCas9 fused to the core catalytic domain of p300 can acetylate target sites in the genome. Acetylation of the target gene synergizes with the action of transcription factors and RNA Polymerase II, resulting in transcriptional upregulation. Adapted from Figure 1b of Zentner et al. (2015). b) Comparison of relative expression levels of IL1RN when dCas9 by itself vs. dCas9 fused to the p300 catalytic core is targeted to the IL1RN promoter. Relative expression levels determined by qRT-PCR. Adapted from Figure 1c of Hilton et al. (2015).

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b. Modulating DNA methylation status with dCas9 systems

i. dCas9-TET1CD — A CRISPR Cas9 system for targeted DNA demethylation

Targeted DNA methylation in mammalian cells predominantly occurs on the fifth carbon of cytosines within CpG dinucleotide sequences. Everything from cell development, differentiation, and tumorigenesis can be regulated by DNA methylation, with hypermethylation an occurrence strongly linked with cancer and neurological diseases (13). Technology that can enable easy modulation of DNA methylation would open up avenues for direct probing of functional relationships between methylation status and gene expression, and even lead to the development of therapies for combating disease.

Designed by Xu et al., dCas9-TET1CD is one of the new technologies capable of editing the epigenome in this way. This system is comprised of a dCas9 fused to the catalytic domain (CD) of TET1 (Ten-eleven translocation methylcytosine dioxygenase 1), an enzyme triggers DNA demethylation (13) (Figure 6a). The accompanying sgRNA can also be modified to include an MS2 dimer that is additionally fused to two more TET1CD modules each(14). Such a system has demonstrated the ability to generate a transcriptional increase in an array of genes in a locus-specific manner, with maximum activation of the RANKL gene observed 4 days post-transfection (14) (Figure 6b). Combined with careful sgRNA design and control of the amounts of the different components, the dCas9-TET1CD system and its accompanying MS2-Tet1CD system is able to effectively demethylate genes at specific locations with little off-target effects and in different human and mouse cell lines.

dCas9-TET1CD’s ability to specifically and easily target the gene(s) of choice will help scientists easily design gene promoter screens for exploration of the functionality of DNA methylation in regulating gene expression in specific genomic contexts, with many potential clinical applications to follow. Recently, the dCas9-TET1CD system in combination with a regular sgRNA was also used to target and perform epigenetic edits on the promoter of BRCA1, a tumor suppressor gene whose over silencing via hypermethylation is associated with nonfamilial breast and ovarian cancers (15). Such a system may also be used to restore functional activity of other tumor suppressor genes essential for the fight against cancer and other diseases.

Figure 6 – The dCas9-TET1CD system for targeted DNA demethylation. a) dCas9 is fused to the catalytic domain of TET1 (TET1CD). The accompanying sgRNA is additionally modified to contain two RNA aptamers that recruit MS2 coat proteins, each fused to a TET1CD. Adapted from Figure 1a of Xu et al. (2016). b) qRT-PCR was used to assess mRNA levels of RANKL gene expression using two sgRNAs designed to target the RANKL promoter region (-800 bp upstream of transcription start site). Adapted from Figure 2c of Xu et al. (2016).

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ii. dCas9-DNMT3A—Methylating DNA via a modified CRISPR Cas9 system

Unlike histone-based control of cell phenotypes, DNA methylation is more stable and long-term in its effects on gene expression. Not only do dCas9-based methylation systems have cross-species capability, they are not sensitive to CpG methylation. This is in contrast to TALE-based systems which are unsuitable for epigenetic manipulation of mammalian promoters due to their CpG methylation sensitivity (16).

dCas9-DNMT3A is the methylating counterpart to the dCas9-Tet1CD system previously discussed. Developed by Vojta et al., this system involves fusion of dCas9 (via a flexible Gly4Ser linker) with the catalytic domain of DNMT3A, an active DNA methyltransferase that is capable of methylating CpG sites in vivo. This dCas9-DNMT3A system successfully induced site-specific CpG methylation upstream and downstream of a BACH2 promoter in HEK293 cells, with the highest concentration of methylation activity (60%) located 27 bp downstream from the PAM sequence (17). This was similarly observed when targeting the IL6ST promoter. Expression levels of both genes were significantly decreased within 10 days after transfection (17). In addition, the simultaneous targeting of multiple gene-specific sites via dCas9-DNMT3A combined with pooled sgRNAs results in a synergistic effect on methylation, boosting methylation by 2x and amplifying the gene silencing power of this system even more (17).

Since its development, the dCas9-DNMT3A has also been used to directly methylate the promoter of the CDKN1A and 2A tumor-suppressor genes(18) whose hypermethylation is correlated with several cancers. The authors of this study observed that methylation of the CDKN1A promoter resulted in decreased expression of CDKN1A and a corresponding increase in the proliferation of the transduced cells (19), further demonstrating the use of this system for functional genomics studies. This construct will have many potential applications for epigenome editing and regulation without causing global alterations in the epigenome.

Figure 6 – The dCas9-DNMT3A system for targeted DNA methylation. a) dCas9 is fused to the catalytic domain of DNMT3A. In vivo, DNMT3A recruits a partner for dimerization along with DNMT3L proteins (shown in dashed red box and ovals, respectively). Adapted from Figure 1a of Vojta et al. (2016). b) RT-qPCR analysis of IL6ST gene expression via dCas9-fused to active DNMT3A. Expression levels induced by dCas9 fused to inactive DNMT3A along with dCas9 accompanied by non-targeting sgRNAs were measured as negative controls. Fold change is relative to mock-transfected cells. Adapted from Figure 4a of Vojta et al. (2016).

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References

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The New State Of The Art: Cas9 For Gene Activation And Repression. La Russa, M. F. and Qi, L. S. 2015, Mol. and Cell. Biol., Vol. 35, pp. 3800-3809.
Genome-Scale Transcriptional Activation By An Engineered CRISPR-Cas9 Complex. Konermann, S., Brigham, M. D., Trevino, A. E., Joung, J., Abudayyeh, O. O., Barcena, C., Hsu, P. D., Habib, N., Gootenberg, J. S., Nishimasu, H., Nureki, O, and Zhang, F. 2015, Nature, Vol. 517, pp. 583-588.
Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology. Jusiak, B., Cleto, S. Perez-Pinera, P., and Lu, T. K. 2016, Trends in Biotech., Vol. 34, pp. 535-547.
CRISPR/Grna-Directed Synergistic Activation Mediator (SAM) Induces Specific, Persistent And Robust Reactivation Of The HIV-1 Latent Reservoirs. Zhang, Y., Yin, C. Zhang, T., Li, F., Yang, W., Kaminski, R., Fagan, P. R., Putatunda, R., Young, W., Khalili, K., and Hu, W. 2015, Sci. Rep., Vol.5, pp. 1-14.
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Epigenome editing by CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers. Hilton, I. B., D’Ippolito, A. M., Vockley, C. M., Thakore, P. I., Crawford, G. E., Reddy, T. E., and Gersbach, C. A. 2015, Nat. Biotechnol., Vol. 33, pp. 510-517.
Functional annotation of native enhancers with a Cas9-histone demthylase fusion. Kearns, N. A., Pham, H., Tabak, B., Genga, R. M., Silverstein, N. J., Garber, M., Maehr, R. 2015, Nature Methods, Vol. 12, pp. 401-403.
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Epigenome editing made easy. Zentner, G. E., and Henikoff, S. 2015, Nature., Vol. 33, pp.606-607.

Introduction

Methods and Tools

sgRNA Design

Screening and Validation

Gene Regulation with dCas9

Gene Knockout/Knock-in Case Studies

Gene Silencing Methods: CRISPR vs. RNAi vs. TALENs

Guide for CRISPR Gene Knockout

CRISPR Crash Course