CRISPR activation

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CRISPR activation (CRISPRa) is a type of CRISPR tool that uses modified versions of CRISPR effectors without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs). [1]

Contents

Like for CRISPR interference, the CRISPR effector is guided to the target by a complementary guide RNA. However, CRISPR activation systems are fused to transcriptional activators to increase expression of genes of interest. Such systems are usable for many purposes including but not limited to, genetic screens and overexpression of proteins of interest. The most commonly-used effector is based on Cas9 (from Type II systems), but other effectors like Cas12a (Type V) have been used as well. [2]

Components

dCas9

Cas9 Endonuclease Dead, also known as dead Cas9 or dCas9, is a mutant form of Cas9 whose endonuclease activity is removed through point mutations in its endonuclease domains. Similar to its unmutated form, dCas9 is used in CRISPR systems along with gRNAs to target specific genes or nucleotides complementary to the gRNA with PAM sequences that allow Cas9 to bind. Cas9 ordinarily has 2 endonuclease domains called the RuvC and HNH domains. The point mutations D10A and H840A change 2 important residues for endonuclease activity that ultimately results in its deactivation. Although dCas9 lacks endonuclease activity, it is still capable of binding to its guide RNA and the DNA strand that is being targeted because such binding is managed by other domains. This alone is often enough to attenuate if not outright block transcription of the targeted gene if the gRNA positions dCas9 in a way that prevents transcriptional factors and RNA polymerase from accessing the DNA. However, this ability to bind DNA can also be exploited for activation since dCas9 has modifiable regions, typically the N and C terminus of the protein, that can be used to attach transcriptional activators. [3]

Guide RNA

See: Guide RNA, CRISPR

A small guide RNA (sgRNA), or gRNA is an RNA with around 20 nucleotides used to direct Cas9 or dCas9 to their targets. gRNAs contain two major regions of importance for CRISPR systems: the scaffold and spacer regions. The spacer region has nucleotides that are complementary to those found on the target genes, often in the promoter region. The scaffold region is responsible for formation of a complex with (d)Cas9. Together, they bind (d)Cas9 and direct it to the gene(s) of interest. Since the spacer region of a gRNA can be modified for any potential sequence, they give CRISPR systems much more flexibility as any genes and nucleotides with a sequence complementary to the spacer region can become possible targets. [3]

Complementary base pairing between the sgRNA and genomic DNA allows targeting of Cas9 or dCas9 Schematic Structure of the Cas9-sgRNA-DNA Ternary Complex.svg
Complementary base pairing between the sgRNA and genomic DNA allows targeting of Cas9 or dCas9

Transcriptional activators

See: Transcriptional Activator, Transcription Factor

Transcriptional Activators are protein domains or whole proteins linked to dCas9 or sgRNAs that assist in the recruitment of important co-factors as well as RNA Polymerase for transcription of the gene(s) targeted by the system. In order for a protein to be made from the gene that encodes it, RNA polymerase must make RNA from the DNA template of the gene during a process called transcription. Transcriptional activators have a DNA binding domain and a domain for activation of transcription. The activation domain can recruit general transcription factors or RNA polymerase to the gene sequence. Activation domains can also function by facilitating transcription by stalled RNA polymerases, and in eukaryotes can act to move nucleosomes on the DNA or modify histones to increase gene expression. [4] These activators can be introduced into the system through attachment to dCas9 or to the sgRNA. Some researchers have noted that the extent of transcriptional upregulation can be modulated by using multiple sites for activator attachment in one experiment and by using different variations and combinations of activators at once in a given experiment or sample. [5] [6] [7]

Expression system

An expression system is required for the introduction of the gRNAs and (d)Cas9 proteins into the cells of interest. Typically employed options include but are not limited to plasmids and viral vectors such as adeno-associated virus (AAV) vector or lentivirus vector.

Specific activation systems

VP64-p65-Rta

The VP64-p65-Rta, or VPR, dCas9 activator was created by modifying an existing dCas9 activator, in which a Vp64 transcriptional activator is joined to the C terminus of dCas9. [1] In the dCas9-VPR protein, the transcription factors p65 and Rta are added to the C terminus of dCas9-Vp64. Therefore, all three transcription factors are targeted to the same gene. The use of three transcription factors, as opposed to solely Vp64, results in increased expression of targeted genes. When different genes were targeted by dCas9, they all showed significantly greater expression with dCas9-VPR than with dCas9-VP64. It has also been demonstrated that dCas9-VPR can be used to increase expression of multiple genes within the same cell by putting multiple sgRNAs into the same cell. [8] dCas9-VPR has been used to activate the neurogenin 2 (link) and neurogenic differentiation 1 (link) genes, resulting in differentiation of induced pluripotent stem cells into induced neurons. [8] A study comparing dCas9 activators found that the VPR, SAM, and Suntag activators worked best with dCas9 to increase gene expression in a variety of fruit fly, mouse, and human cell types. [9]

The dCas9-VPR activator increases transcription at the gene that it targets. DCas9-VPR activator.svg
The dCas9-VPR activator increases transcription at the gene that it targets.

Synergistic activation mediator

To overcome the limitation of the dCas9-VP64 gene activation system, the dCas9-SAM system was developed to incorporate multiple transcriptional factors. Utilizing MS2, p65, and HSF1 proteins, dCas9-SAM system recruits various transcriptional factors working synergistically to activate the gene of interest.

The dCas-SAM system uses msgRNA that has attached aptamers for different transcriptional factors (MS2,p65 and HSF1) to bind. DCas SAM system.png
The dCas-SAM system uses msgRNA that has attached aptamers for different transcriptional factors (MS2,p65 and HSF1) to bind.

In order to assemble different transcriptional activators, the dCas9-SAM system uses a modified single guide RNA (sgRNA) that has binding sites for the MS2 protein. Hairpin aptamers are attached to the tetra loop and the stem loop 2 of the sgRNA to become binding sites for dimerized MS2 bacteriophage coat proteins. As the hairpins are exposed outside of the dCas9-sgRNA complex, other transcriptional factors can bind to the MS2 protein without disrupting the dCas9-sgRNA complex. Thus, the MS2 protein is engineered to include p65 and HSF1 proteins. The MS2-p65-HSF1 fusion protein interacts with the dCas9-VP64 to recruit more transcriptional factors onto the promoter of the target genes.

Employing the dCas-SAM system, Zhang et al. (2015) successfully reactivated the latent HIV gene to over-express viral proteins from the HIV host cells. [10] They were able to over-express viral proteins substantially to trigger apoptosis of HIV-1 latent cells due to the toxicity of viral proteins. In another dCas-SAM system experiment, Konermann et al. (2015) found genes in melanoma cells that give resistance to a BRAF inhibitor through activating candidate genes via dCas system. [7] Thus, the dCas9-SAM system can further be employed to activate latent genes, develop gene therapies, and discover new genes.

SunTag

The SunTag activator system uses the dCas9 protein, which is modified to be linked with the SunTag. The SunTag is a repeating polypeptide array that can recruit multiple copies of antibodies. Through attaching transcriptional factors on the antibodies, the SunTag dCas9 activating complex amplifies its recruitment of transcriptional factors. In order to guide the dCas9 protein to its target gene, the dCas9 SunTag system uses sgRNA.

Tanenbaum et al.(2014) are credited for creating the dCas9 SunTag system. [11] For the antibodies, they employed GCN4 antibodies which was bound to transcriptional factor VP64. In order to transport the antibodies to the nuclei of the cells, they attached NLS tag. To confirm the nuclear localization of the antibodies, sfGFP was used for visualization purpose. Therefore, the GCN4-sfGFP-NLS-VP64 protein was developed to be interact with dCas SunTag system. The antibodies successfully bound to SunTag polypeptides and activated target CXCR4 gene in K562 cell lines. [11] Comparing with the dCas9-VP64 activation complex, they were able to increase the CXCR4 gene expression 5-25 times greater in K562 cell lines. Not only was there a greater CXCR4 protein overexpression but also CXCR4 proteins were active to further travel on the transwell migration assay. Thus, the dCas9-SunTag system can be used to activate genes that are present latently such as virus genes.

The use of the Suntag system allows multiple antibodies fused to VP64 to bind to dCas9-Suntag. That in turn recruits RNA polymerase and increases gene expression. DCas9 Suntag Activator (2).jpg
The use of the Suntag system allows multiple antibodies fused to VP64 to bind to dCas9-Suntag. That in turn recruits RNA polymerase and increases gene expression.

Applications

The dCas9 activation system allows a desired gene or multiple genes in the same cell to be expressed. It is possible to study genes involved in a certain process using a genome wide screen that involves activating expression of genes. Examining which sgRNAs yield a phenotype suggests which genes are involved in a specific pathway. The dCas9 activation system can be used to control exactly which cells are activated and at what time activation occurs. dCas9 constructs have been made that turn on a dCas9-activator fusion protein in the presence of light or chemicals. Cells can also be reprogrammed or differentiated from one cell type into another by increasing the expression of certain genes important for the formation or maintenance of a cell type. [12]

Greater control over gene expression

One research group used a system in which dCas9 was fused to a particular domain, C1B1. When blue light is shined on the cell, the cryptochrome 2 (Cry2) domain binds to C1B1. The Cry2 domain is fused to a transcriptional activator, so blue light targets the activator to the spot where dCas9 is bound. The use of light allows a great deal of control over when the targeted gene is activated. Removing the light from the cell results in only dCas9 remaining at the target gene, so expression is not increased. In this way, the system is reversible. [13] A similar system was developed using chemical control. In this system, dCas9 recruits an MS2 fusion protein that contains the domain FKBP. In the presence of the chemical RAP, an FRB domain fused to a chromatin modifying complex binds to FKBP. Whenever RAP is added to the cells, a specific chromatin modifier complex can be targeted to the gene. That allows scientists to examine how specific chromatin modifications affect the expression of a gene. [14] The dCAs9-VPR system is used as an activator by targeting it to the promoter of a gene upstream of the coding region. A study used various sgRNAs to target different portions of the gene, finding that the dCas9-VPR activator can act as an activator or a repressor, depending on the location it binds. In a cell, sgRNAs targeting the promoter could allow dCas9-VPR to increase expression, while sgRNAs targeting the coding region of the gene result in dCas9-VPR decreasing expression. [15]

Genome wide activation

The versatility of sgRNAs allows dCas9 activators to increase the expression of any gene within an organism's genome. That could be used to increase expression of a protein coding gene or a transcribed RNA. A paper demonstrated that genome wide activation could be used to determine which proteins are involved in mediated resistance to a specific drug. [7] Another paper used genome wide activation of long, noncoding RNAs and observed that increasing the expression of certain long noncoding RNAs conferred resistance to the drug vemurafenib. [16] In both cases, the cells that survive the drug could be studied to determine which sgRNAs they contain. That allows researchers to determine which gene was activated in each surviving cell, which suggests which genes are important for resistance to that drug.

Use in organisms

A dCas9 fusion with VP64, p65, and HSF1 (heat shock factor 1) allowed researchers to target genes in Arabidopsis thaliana and increase transcription to a similar level as when the gene itself is inserted into the plant's genome. For one of the two genes tested, the dCas9 activator changes the number and size of leaves and made the plants better able to handle drought. The authors conclude that the dCas9 activator can create phenotypes in plants that are similar to those observed when a transgene is inserted for overexpression. [17] Researchers have used multiple guide RNAs to target dCas9 activation system to multiple genes in a specific mouse strain in which dCas9 can be turned on in specific cell lines using the Cre recombinase system. Scientists used the targeting and increased expression of several genes to examine the processes involved in regeneration and carcinomas of the liver. [18]

Related Research Articles

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

<span class="mw-page-title-main">Functional genomics</span> Field of molecular biology

Functional genomics is a field of molecular biology that attempts to describe gene functions and interactions. Functional genomics make use of the vast data generated by genomic and transcriptomic projects. Functional genomics focuses on the dynamic aspects such as gene transcription, translation, regulation of gene expression and protein–protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures. A key characteristic of functional genomics studies is their genome-wide approach to these questions, generally involving high-throughput methods rather than a more traditional "candidate-gene" approach.

<span class="mw-page-title-main">CRISPR</span> Family of DNA sequence found in prokaryotic organisms

CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes and provide a form of acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.

A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence or have a general affinity to DNA. Some DNA-binding domains may also include nucleic acids in their folded structure.

Guide RNA (gRNA) or single guide RNA (sgRNA) is a short sequence of RNA that functions as a guide for the Cas9-endonuclease or other Cas-proteins that cut the double-stranded DNA and thereby can be used for gene editing. In bacteria and archaea, gRNAs are a part of the CRISPR-Cas system that serves as an adaptive immune defense that protects the organism from viruses. Here the short gRNAs serve as detectors of foreign DNA and direct the Cas-enzymes that degrades the foreign nucleic acid.

<span class="mw-page-title-main">Artificial transcription factor</span>

Artificial transcription factors (ATFs) are engineered individual or multi molecule transcription factors that either activate or repress gene transcription (biology).

<span class="mw-page-title-main">Insert (molecular biology)</span>

In Molecular biology, an insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism.

<span class="mw-page-title-main">Genome editing</span> Type of genetic engineering

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

<span class="mw-page-title-main">CRISPR interference</span> Genetic perturbation technique

CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna. Sequence-specific activation of gene expression refers to CRISPR activation (CRISPRa).

<span class="mw-page-title-main">Epigenome editing</span>

Epigenome editing or epigenome engineering is a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites. Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.

A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.

<span class="mw-page-title-main">Cas12a</span> DNA-editing technology

Cas12a is a subtype of Cas12 proteins and an RNA-guided endonuclease that forms part of the CRISPR system in some bacteria and archaea. It originates as part of a bacterial immune mechanism, where it serves to destroy the genetic material of viruses and thus protect the cell and colony from viral infection. Cas12a and other CRISPR associated endonucleases use an RNA to target nucleic acid in a specific and programmable matter. In the organisms from which it originates, this guide RNA is a copy of a piece of foreign nucleic acid that previously infected the cell.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

Perturb-seq refers to a high-throughput method of performing single cell RNA sequencing (scRNA-seq) on pooled genetic perturbation screens. Perturb-seq combines multiplexed CRISPR mediated gene inactivations with single cell RNA sequencing to assess comprehensive gene expression phenotypes for each perturbation. Inferring a gene’s function by applying genetic perturbations to knock down or knock out a gene and studying the resulting phenotype is known as reverse genetics. Perturb-seq is a reverse genetics approach that allows for the investigation of phenotypes at the level of the transcriptome, to elucidate gene functions in many cells, in a massively parallel fashion.

CRISPR-Display (CRISP-Disp) is a modification of the CRISPR/Cas9 system for genome editing. The CRISPR/Cas9 system uses a short guide RNA (sgRNA) sequence to direct a Streptococcus pyogenes Cas9 nuclease, acting as a programmable DNA binding protein, to cleave DNA at a site of interest.

Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave, creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.

<span class="mw-page-title-main">CRISPR gene editing</span> Gene editing method

CRISPR gene editing is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.

<span class="mw-page-title-main">Genome-wide CRISPR-Cas9 knockout screens</span> Research tool in genomics

Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations. The approach utilises the CRISPR-Cas9 gene editing system, coupled with libraries of single guide RNAs (sgRNAs), which are designed to target every gene in the genome. Over recent years, the genome-wide CRISPR screen has emerged as a powerful tool for performing large-scale loss-of-function screens, with low noise, high knockout efficiency and minimal off-target effects.

<span class="mw-page-title-main">GESTALT</span> Method for lineage tracing using CRISPR-Cas9-edited barcodes

Genome editing of synthetic target arrays for lineage tracing (GESTALT) is a method used to determine the developmental lineages of cells in multicellular systems. GESTALT involves introducing a small DNA barcode that contains regularly spaced CRISPR/Cas9 target sites into the genomes of progenitor cells. Alongside the barcode, Cas9 and sgRNA are introduced into the cells. Mutations in the barcode accumulate during the course of cell divisions and the unique combination of mutations in a cell's barcode can be determined by DNA or RNA sequencing to link it to a developmental lineage.

References

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  10. ↑ Zhang Y, Yin C, Zhang T, Li F, Yang W, Kaminski R, et al. (November 2015). "CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs". Scientific Reports. 5 (1): 16277. Bibcode:2015NatSR...516277Z. doi:10.1038/srep16277. PMC   4633726 . PMID   26538064.
  11. 1 2 Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD (October 2014). "A protein-tagging system for signal amplification in gene expression and fluorescence imaging". Cell. 159 (3): 635–46. doi:10.1016/j.cell.2014.09.039. PMC   4252608 . PMID   25307933.
  12. ↑ Dominguez AA, Lim WA, Qi LS (January 2016). "Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation". Nature Reviews. Molecular Cell Biology. 17 (1): 5–15. doi:10.1038/nrm.2015.2. PMC   4922510 . PMID   26670017.
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