Introduction: Schizophrenia is a complex biological disorder with multifactorial mode of transmission where non-genetic determinants are also play important role. It is now clear that it involves combined effect of many genes, each conferring a small increase in liability to the illness. Thus no causal disease genes or single gene of major effects, only susceptible genes are operating. Given this complexity, it comes as no surprise of the difficulty to find susceptible genes. However, schizophrenia genes have been found at last. Recent studies on molecular genetics of schizophrenia which focused on positional and functional candidate genes postulated to be associated with schizophrenia are beginning to produce findings of great interest (1). Schizophrenia is a heterogeneous syndrome, affecting ~1% of the population and characterized by debilitating positive, negative, and cognitive symptoms in addition to severe comorbidities. Despite the enormous burden on worldwide health including 1.9–2.8% of total years lived with disability and a 10–20 year reduction in life expectancy, no drugs with novel mechanisms of action have emerged in the last three decades. Current antipsychotic medications only achieve full symptom remission in 15–25% of affected individuals and adverse side-effects such as weight gain, metabolic disturbances, over-sedation, extrapyramidal symptoms, and agranulocytosis are persistent problems (2). These include neuregulin (NRG-1, 8p12–21), dysbindin, (DTNBP1,6p22.3), G72 (13q34), D-amino acid oxidase (DAAO,12q24), proline dehydrogenase (PRODH-2, 22q11.21), catechol-O-methyltransferase (COMT, 22q11.21), regulator of G protein signaling (RGS-4), 5HT2A and dopamine D3 receptor (DRD3)(1).
We have investigated the gene for dystrobrevin-binding protein 1 (DTNBP1), or dysbindin, which has been strongly suggested as a positional candidate gene for schizophrenia, in three samples of subjects with schizophrenia and unaffected control subjects of German (418 cases, 285 controls), Polish (294 cases, 113 controls), and Swedish (142 cases, 272 controls) descent. We analyzed five single-nucleotide polymorphisms (P1635, P1325, P1320, P1757, and P1578) and identified significant evidence of association in the Swedish sample but not in those from Germany or Poland. The results in the Swedish sample became even more significant after a separate analysis of those cases with a positive family history of schizophrenia, in whom the five-marker haplotype A-C-A-T-T showed a P value of .00009 (3.1% in controls, 17.8% in cases; OR 6.75; P=.00153 after Bonferroni correction). Our results suggest that genetic variation in the dysbindin gene is particularly involved in the development of schizophrenia in cases with a familial loading of the disease (3). DTNBP1 (dystrobrevin binding protein 1) is a leading candidate susceptibility gene in schizophrenia and is associated with working memory capacity in normal subjects. In schizophrenia, the encoded protein dystrobrevin-binding protein 1 (dysbindin-1) is often reduced in excitatory cortical limbic synapses. We found that reduced dysbindin-1 in mice yielded deficits in auditory-evoked response adaptation, prepulse inhibition of startle, and evoked γ-activity, similar to patterns in schizophrenia. In contrast to the role of dysbindin-1 in glutamatergic transmission, γ-band abnormalities in schizophrenia are most often attributed to disrupted inhibition and reductions in parvalbumin-positive interneuron (PV cell) activity. To determine the mechanism underlying electrophysiological deficits related to reduced dysbindin-1 and the potential role of PV cells, we examined PV cell immunoreactivity and measured changes in net circuit activity using voltage-sensitive dye imaging. The dominant circuit impact of reduced dysbindin-1 was impaired inhibition, and PV cell immunoreactivity was reduced. Thus, this model provides a link between a validated candidate gene and an auditory endophenotypes. Furthermore, these data implicate reduced fast-phasic inhibition as a common underlying mechanism of schizophrenia-associated intermediate phenotypes (4).
Fig.1. Mice with reduced dysbindin-1 expression (Dys−/−) show a prominent loss of inhibition. During imaging of membrane voltage in the hippocampal area CA1 (A), electrical stimulation generates a brief local excitation (red) followed by a hyperpolarization (blue; B). In Dys−/− mice, the dominant impact is loss of inhibition (C). Decay time constant of this response was used to measure the kinetics of repolarization (C Inset). (D) The decay time constant was significantly prolonged in Dys1−/− mice. (E) When the decay time constant from Dys−/− mice is plotted over sigmodal waves representing 80 or 30 Hz, it is clear that it would be more difficult for the CA1 neurons in the Dys−/− mice to generate higher-frequency oscillations (red trace) compared with normal mice (blue trace). Thus, the loss of high γ-activity (60–100 Hz) observed in the Dys−/− mice and characteristic of schizophrenia may be directly caused by reduced fast inhibitory function (4).
CRISPR/Cas9 Technology
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems and their CRISPR associated proteins (Cas proteins) have allowed for an unprecedented ability to manipulate the genome. Key amongst the applications of these systems is their use in gene editing for targeted gene knockout, knockin, and modification (6). It makes it possible to correct errors in the genome and turn on or off genes in cells and organisms quickly, cheaply and with relative ease. It has a number of laboratory applications including rapid generation of cellular and animal models, functional genomic screens and live imaging of the cellular genome (7). CRISPR systems exist across a wide range of bacterial species, providing a rich source of functional diversity for genome editing in eukaryotic cells (8,9). The first described, and most commonly used, is the type-II CRISPR-Cas9 system from Streptococcus pyogenes (SpCas9), a DNA endonuclease that is directed to induce double strand breaks (DSBs) at specific genomic loci via a programmable guide RNA (gRNA) molecule that mediates complementary DNA-RNA base pairing. For SpCas9 to efficiently bind and cleave DNA, the target sequence must be flanked on the 3′ side by an ‘NGG’ protospacer adjacent motif (PAM) sequence (10). Since the initial implementation of CRISPR systems in eukaryotic cells there has been a rapid expansion of variant enzymes that broaden the capabilities of CRISPR-based platforms (9,11). Each has its own set of features and criteria for sequence recognition that provides added flexibility for adaptation as a research or therapeutic tool (12) that are smaller than SpCas9, allowing easier packaging into size-limited delivery vectors such as adenoassociated virus (AAV) (13), while others can catalyse the maturation of their own gRNAs, simplifying the process of target multiplexing (14). Yet another family of Cas enzymes named Cas13 (previously known as C2c2) target RNA instead of DNA, providing an alternative approach to manipulate gene expression (15,16).
The Cas9 protein is an endonuclease containing two nuclease domains, RuvC and HNH. The RuvC domain cleaves noncomplementary DNA strands, while the HNH domain cleaves complementary DNA strands. The sgRNA is composed of the trans-activating crRNA (tracrRNA) and crRNA. The crRNA contains a 20-nt protospacer element and an additional sequence that is complementary to the tracrRNA. The tracrRNA hybridizes to the crRNA and binds the Cas9 protein, forming the CRISPR-Cas9/sgRNA complex to create double-stranded breaks (DSBs) at target sites in the genome. The dual-tracrRNA:crRNA is normally engineered as a single-strand sgRNA containing two crucial segments: a duplex RNA structure at the 3′ end to bind Cas9 and a guide sequence at the 5′ end to bind target DNA sequence. this two-component system is simple but powerful. sgRNA recognizes a specific sequence in the genome, and Cas9 acts as a pair of scissors to cleave the DNA sequence (17). A number of challenges remain before the potential of CRISPR/Cas9 can be translated to effective treatments at the bedside. A particular issue is how to deliver gene editing to the right cells, especially if the treatment is to be delivered in vivo. To safely deliver Cas9 encoding genes and guide RNAs in vivo without any associated toxicity, a suitable vector is needed. A smaller Cas9 gene could be used, but this has additional implications on efficacy (7).
Fig. 2. Schematic of the RNA-guided Cas9; The Cas9 nuclease from S. pyogenes (in yellow) is targeted to genomic DNA by an sgRNA consisting of a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence pairs with the DNA target (blue bar on top strand), directly upstream of a requisite 5′-NGG adjacent motif (PAM; pink). Cas9 mediates a DSB ~3 bp upstream of the PAM (red triangle) (18,19).
technologies As with other designer nuclease technologies such as ZFNs and TALENs, Cas9 can facilitate targeted DNA DSBs at specific loci of interest in the mammalian genome and stimulate genome editing via NHEJ or HDR. Cas9 offers several potential advantages over ZFNs and TALENs, including the ease of customization, higher targeting efficiency and the ability to facilitate multiplex genome editing. Cas9 can be easily retargeted to new DNA sequences by simply purchasing a pair of oligos encoding the 20-nt guide sequence. In contrast, retargeting of TALEN for a new DNA sequence requires the construction of two new TALEN genes. Although a variety of protocols exist for TALEN construction, it takes substantially more hands -on time to construct a new pair of TALENs. Mutating catalytic residues in either the RuvC or the HNH nuclease domain of SpCas9 converts the enzyme into a DNA nicking enzyme. In contrast, TALENs cleave nonspecifically in the 12–24-bp linker between the pair of TALEN monomer-binding sites (18).
dCas9-KRAB Repression
The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it has been difficult to assay the function of single transcription factor binding sites within larger transcription networks. CRISPR interference/activation (CRISPRi/a) technology provides a simple and efficient approach for targeted repression or activation of gene expression in the mammalian genome. It is highly flexible and programmable, using an RNA-guided nuclease-deficient Cas9 (dCas9) protein fused with transcriptional regulators for targeting specific genes to effect their regulation. Multiple studies have shown how this method is an effective way to achieve efficient and specific transcriptional repression or activation of single or multiple genes. Sustained transcriptional modulation can be obtained by stable expression of CRISPR components, which enables directed reprogramming of cell fate. Here, we introduce the basics of CRISPRi/a technology for genome repression or activation (A).
The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Here we used a strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Dystrobrevin Binding Protein 1 (DTNBP1) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system (5).
Methods: 10 Oligonucleotides (gRNAs) were designed with Chop-Chop, and we're analized with data's to check the specificity and off Target activity. The gRNA which showed more efficiency results, was further investigated. With the help of the gRNA webserver tool, we were able to show that the selected gRNA has the highest effectiveness rate while the lowest off-target rate is reported. Then, this gRNA was compared to CRISPR/dCas9-KRAB (7540bp) plasmid of AddGene tool to be identified as efficient DTNBP1 gene silencing Complex.
Results: 10 top gRNAs designed for DTNBP1 Gene silencing with Chop-Chop tool. Throughout the analysis, tge Ranked 1 oligonucleotide had been selected for CRISPR complex. List of possible off-targets of our Ranked 1 oligonucleotide. According to further details and analysis, the Highest level of specificity have been reported
Conclusion: The corresponding gRNA with maximum effect and on-target was selected with the help of data analysis. Next, the plasmid related to CRISPR/dCas9-KRAB was selected to target the DTNBP1 gene with the help of AddGene's reviewed results. At the end, gRNA was analyzed by gRNA webserver tool regarding the on/off target ability and Specificity, and finally, the highest level of effectiveness was shown