Are you still thinking that CRISPR-Cas system is only used for cutting DNA in fragments to generate knock out models? If you answered ‘yes’, then join us in a quick trip through a new landscape of applications and opportunities.
Nowadays, when talking about CRISPR-Cas systems, most people still think about gene editing, knock-out/in (KO/KI) models, point mutation delivery and genetic modified organisms. Even though the CRISPR-Cas system was primarily adapted for gene editing, in recent years the system has evolved, allowing scientists to use CRISPR-Cas tools to study biological processes without altering the DNA sequence. Most of the non-editing CRISPR-based technologies rely on a Cas9 nuclease variant called ‘death Cas9’ (dCas9), which is a mutant form of the nuclease whose endonuclease activity has been removed through point mutations (D10A and H840A). As a consequence, dCas9 lacks endonuclease activity but is still capable to bind to its guide RNA (gRNA) and the DNA strand that is being targeted.
Multiples tools based on the same molecule
Editing histone epigenetic marks: Histones are involved in transcriptional regulation. Silenced chromatin is characterized by H3K9me3 and H3K27me3, whereas active chromatin is characterized by H3K4me/H3K79me and H3K9ac/H3K27ac. Targeted modifications of epigenetics in regulatory regions or in-site recruitment of transcriptional factors is the basis of CRISPR-interference (CRISPR-i) and CRISPR-activation (CRISPR-a) approaches. In CRISPR-i, the dCas9 is fused to a KRAB transcriptional repressor which attracts histone deacetylases and methyltransferases that add epigenetic marks of inactive heterochromatin. CRISPR-a take advantage of dCas9 proteins to recruit activation domains to regulatory genomic elements and induce target gene transcription. CRISPR-a functions by acetylating histones in target regions or directly activating genes by recruiting transcription factors. The dCas9 can be fused with p300, p65, p65/HSF1 or VP(16)n [1-5]. Another implementation consists of drawing an active cytomegalovirus promoter to the target to activate the region of interest.
Regulating the epigenome represents a promising strategy for treating diseases by modulating histone marks and transcription factor rather than editing the DNA sequences. CRISPR-a/i systems induceepigenetic remodeling by recruiting the transcriptional machinery, altering gene expression in vivo to generate physiologically relevant phenotypes without altering the DNA. These systems can be used to transcriptionally activate either single genes or combinations of genes, including extremely large genes. Furthermore, we can express genes to compensate for disease-associated genetic mutations, to overexpress long non-coding RNAs or GC-rich genes to reveal their biological functions. CRISPR-a/i tools can be applied to the study of signaling pathways in different cell subsets, analyzing the loss of function effect of key genes for cellular processes, for the identification of regulatory regions near the promoters, and even for the reversible knock-down of target genes.
Epigenetic remodeling: Activity of genes is mainly determined by chromatin architecture in regulatory regions, so epigenetically modifying the DNA or histones will alter chromatin state. dCas9 can be designed to directly modify DNA epigenetics by methylation or demethylation, as well as to change epigenetic marks or histones by acetylation/deacetylation, methylation/demethylation, or recruitment of transcription factors. dCas9-DNMT3A-3L recruitment can lead to a methylation of a genome region of interest [6-8], whereas recruitment of dCas9-TET1 efficiently demethylates up to 90% of target DNA regions [9, 10].
CRISPR as a remodeler of tridimensional chromatin structure: Although distal regulatory elements are located many thousands of nucleotides away from gene promoters, they strongly impact gene transcription when drawn close together. To understand many physiological and pathological processes, we must identify and elucidate the function of these distal regulatory elements. A method called CLOuD9 has been developed, relying on the interaction between two orthologous dCas9 fused to dimerizing domains PYL1 and ABI1. One dCas9 interacts with the distal region, while the other targets the promoter of interest. Then, an inducer compound promotes que dimerization of the dCas9 proteins, thus favoring the interaction of the bound chromosomal regions. CLOuD9 can be devised to directly manipulate the 3D architecture of chromatin, analyze distal regulatory regions and define new intra- and inter-chromosomal links .
Brezgin et al. Dead Cas Systems: Types, Principles and Applications. Int. J. Mol. Sci. 2019, 20, 6041; doi:10.3390/ijms20236041
Fluorescent DNA tagging using dCas9: dCas9 fusion with fluorescent molecules such as eGFP allows the visualization of the spatio-temporal organization of the genome, prompting new insights of how chromatin structure and function are intertwined [12-15]. In situ hybridization (FISH) is a powerful tool to physically map DNA sequences for research and diagnostic purposes; nevertheless, it has major drawbacks as the need of any kind of DNA denaturation to achieve complementary base pairing in dsDNA, a treatment that degrades the DNA structure. Furthermore, FISH includes hybridization and post hybridization washes, taking hours to achieve the final result. In contrast, CRISPR-Cas9-mediated in situ labelling of genomic loci does not require DNA denaturation, allowing a better structural preservation of the target DNA. RGEN-ISL (‘RNA-guided endonuclease – in situ labelling’) uses a fluorophore coupled sgRNA and a dCas9, allowing the labelling of DNA elements in fixed mammalian cells. RGEN-ISL preserves the natural spatio-temporal organization of the chromatin and allow the detection of multicolored genomic sequences in a specific and simultaneous manner.
In addition to RGEN-ISL, another method called CRISPRainbow allows labelling the DNA based on a dCas9 combined with engineered sgRNAs scaffolds that bindsets of fluorescent proteins. CRISPRainbow can image up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci .
DNA purification with CRISPR: Epitope tagged dCas9 can also be used to immunoprecipitate and purify a genomic locus with its associated proteins or RNA in a process called enChIP [16-19]. To understand the molecular mechanisms underlying the genome functions, it is essential to identify the molecules associated with genomic regions of interest . Chromosome conformation capture (3C) and its derivatives (4C, Hi-C) can be used to detect interactions between genomic regions, and other techniques as proteomic of isolated chromatin (PICh) can be used to identify proteins associated with multicopy loci. The enChIP technique was developed for locus specific biochemical analysis of genome functions such as transcription and epigenetic regulation. enChIP takes advantage of the CRISPR-Cas system for the isolation of specific genomic regions, as follows:
- A ribonucleoprotein (RNP) complex comprising the specific gRNA and the dCas9 is generated.
- The RNP can be fused with a tag and the fusion protein is expressed in the cell or organism to be analyzed.
- Cells are crosslinked and the chromatin fraction is isolated and fragmented by sonication or digestion with endonucleases.
- Chromatin complexes containing the RNP are affinity-purified.
- Isolated chromatin complexes are reverse-crosslinked and proteins are identified by MS, whereas RNA and DNA are identified by NGS or microarray analysis.
Moreover, alternative methods have been developed to tag endogenous proteins, thus enabling specific isolation without the need for custom antibodies [21, 22].
CRISPR tools applied to diagnostic procedures: CRISPR-Cas technology is not only constrained to what is called ‘basic science’, but also rapidly and accurately speeds up the development of infectious disease diagnostics. The CRISPR/Cas9-based tools were firstly used to detect Zika virus in 2016 with a method that combined CRISPR-Cas9 with NASBA isothermal amplification  and methicillin-resistant Staphylococcus aureus in 2017 combining the nuclease with FISH . Combination of CRISPR-Cas9 with optical DNA mapping was also applied to identify bacterial antibiotic resistance genes .
A technique called DETECTR (DNA Endonuclease-targeted CRISPR Trans Reporter) was developed by Chen et al.  and provide attomolar sensitivity for DNA detection, stablishing a straightforward platform for molecular diagnostics.
Another platform called SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) and based on CRISPR-type VI Cas13 was developed in 2017. The method integrates isothermal recombinase polymerase amplification (RPA) or reverse transcription RPA with the nuclease activity of the Cas13 . SHERLOCK allowed for the discrimination of targets that differed only a single nucleotide.
As we can see, CRISPR-Cas system arises as a promising technique with a broad range of applications, from basic science to diagnostics. Furthermore, this technique has been also applied for industrial purposes, creating a landscape of growing versatility.
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