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Discussion

The manipulation of the transcription and post-transcription process has been under investigation for decades and the ability to manipulate various genes have been analysed recently, through various technological interventions. The utilisation of the CRISPR technology for the transcriptional regulation process has been investigated lately and includes the synthetic transcription of the various factors responsible for the manipulation of transcription mechanism. CRISPR or clustered regularly interspaced short palindromic repeats is the mechanism utilised to alter various genes in the animal, plant and bacterial models. The knocking out of the particular genes in these models, leads researchers to identify which traits or behaviours are affected by the removal of the selected gene. The CRISPR associated Cas system is utilised due to their ability to exploit the complementarity between the gRNA (guide RNA) and the target sequence which is mediates he sequence specific target cleavage hence qualifying as a programmable editing tool (gene editing tool) (Pickar-Oliver & Gersbach, 2019). The utilisation of the CRISPR technology for transcriptional regulation has thus been beneficial in many aspects and this research review will include the analysis of four research articles and 3 review articles. The development of the transcriptional regulators for the gene editing process has thus been researched in latest years.

The research conducted by Andriy et al., (2016) was based on the transcriptional regulation of the CRISPR-Cas9 principles, advances and applications. The research has been based with consideration as CRISPR Cas 9 system to be a promising system for the genome editing process, as well as the transcriptome and epigenome perturbation. The CRISPR Cas 9 presents proper ease of use and increased modular as well as programmable nature of the mechanism leads to its widespread usage of the mechanism in transcriptional inhibition, DNA localisation imaging and genetic screening. The review paper presented the non-editing presentation of the CRISPR Cas 9 mechanism for the metabolic engineering, transcriptome perturbation and synthetic biology. The paper presents transcriptional regulation with the CRISPR Cas 9 mechanism and the direction of the Cas 9 to its targets. The review article presents the transcriptional regulation presented by the CRISPR Cas 9 mechanism and the relatable gene circuits and synthetic promoters have been extensively discussed. The applications of the transcriptional regulation has also been elaborated thoroughly and thus the importance of the same have been presented in proper details. The dCas 9 system has been considered as a useful tool for the biotechnological applications, as the explicit demonstration of the traditional RNAi has been in usage since many years. The multiple sgRNA has been concurrently being utilised for the extension of the screening results as it helps the testing of the variant strains with multiple transcriptional perturbations (Zhu, Li & Gao, 2020). The authors in this article have been presenting the approaches and the concurrent down regulation as well as up regulation of the enzymes which enables the quick construction of various metabolic variant pathways. The review thus demonstrated the emergence of the regulation of transcription process and the generation of the novel gene regulatory circuits which cause the efficient production of the cell types and compounds.

Transcriptional regulation with CRISPR Cas 9 mechanism

In the review article published by Sander & Young (2014), the authors have demonstrated the targeted genome editing which is utilised by researchers as a mainstream technology method. The adoption of such technology has mostly been due to the emergence of CRISPR technology which utilises the RNA guided nuclease Cas 9. The utilisation is mostly due to the customisation property of this nuclease. The modification of endogenous varieties of genes has thus enhanced its applicability and effectively. The modified CRISPR Cas 9 system has been adequately demonstrated by the authors in this article which has specifically been modified accordingly for recruiting the heterologous domains which will regulate the endogenous gene expression in living cells. The alteration of the gene expression and genome sequence has thus been of immense importance in this method. The authors have demonstrated the property of many bacteria to properly employ the system of CRISPR to protect themselves against invading nucleic acids and hence provide self-immunisation. The sequences of the invading DNA are interspersed with CRISPR repeat sequences which are expressed as arrays, especially within the bacterial host genome. The type II CRISPR system which is obtained from S. pyogene, is included for inducing the specific DSBs and target the genome editing. The property of the Cas 9 system to direct any DNA sequence from the N20-NGG by altering the initial 20 nucleotides of the gRNA, is thus utilised (Choi & Meyerson, 2014). The article presents details about the RGNs cleaving the target sites but the question remains as to the frequency of these targeted cleavages. The off-target sites as identified by researchers in human cells are different from onto six locations. The detection of these locations are possible through the utilisation of the T7 endonuclease which is sent through mutation mismatch tests due to their sensitivity.

In the article published by Mahas et al. (2018), the authors have discussed the harnessing of the CRISPR Cas system for the programmable transcriptional as well as post-transcriptional regulation. The CRISPR Cas system is facilitated by the prokaryotes as adaptive molecular immunity mechanism for defending against any invading nucleic acids. The two components of the Cas 9 system as well as the importance of the combination with the sgRNA which has been identified as one of the most potent genome editing owing to its efficacy and simplicity. The control of the gene expression at both the transcriptional levels and post-transcriptional levels, has been a safe approach, especially in the therapeutic and clinical setting. The Cas 9 is effective in inactive mutations in the HNH and RuvC in the nuclease deficient dCas9. The transcriptome and genome alteration capability of the Cas system has thus been employed in settings beyond the various DNA targeting mechanisms. The genome engineering, according to these authors, has been democratised by the CRISPR/Cas system due to the substantial disadvantages of the protein genome editing methodology. The authors have identified the necessity to advance and produce libraries which are based on the RNA binding protein capabilities, while eliminating the various programmable RNA targeting proteins, but these have to be conformed to the nuclei acids which are complementary. Recent studies and researches, as identified by the authors, have been mostly focused on identifying and enabling the genome editing process and specifically for transcriptional regulation (Mojica & Montoliu, 2016). The authors have discussed the various modalities for the targeted RNA interferences which have been based on potential application of the CRISPER Cas 9 system as they are programmable transcriptional regulators with broad applicability including synthetic biological applications, functional biology and biotechnology.

CRISPR Cas 9 system for targeted genome editing

The article published by Genga et al., (2016), has been based on the control of transcription of the human pleuripotent cells while utilising the CRISPR effectors. The authors have elaborated that the ability to operate the various transcription mechanisms in the human pleuripotent stem cells or hPSCs have been a fundamental discovery for the understanding of the various cellular state and differentiation mechanisms of the human cells. The CRISPR system has been providing a methodical approach for the rapidly testing gene function in the mammalian cells which includes the human pleuripotent stem cells. The authors have described an application of the CRISPR effector medicates transcriptional regulation in the hPSCs by properly targeting a synthetic promoter which drives the GFP transgene and the demonstration also includes the effectiveness and ease of the CRISPR effector mediated transcriptional regulation in the hPSCs. The CRISPR mechanism has been identified and suppression of the foreign DNA has been RNA guided bacterial immune responses. The mammalian systems has been influenced through the type II CRISPR system which has been recently adopted for the gene editing process. The combination of the dCas9 system with the effector domains have been mostly based on the site specific programmable system (Burstein et al., 2017). The dCas9 has been identified to be localised to the various genomic location which is involved with the steric obstruction of various polymerase recruitment as well as transcriptional elongation. This causes the transcriptional suppression and the dissection of the specific contribution of the components which maintains the biological state has been identified as the system which inhibits the gene function (Zhang et al., 2019). In the hPSCs, the utilisation of the CRISPR Cas 9 system has been allowing the identification of various genes and processes which controls the cellular state. The GFP expression in various cells have been identified to be affected by the CAG receptors.

The article presented by Lowder et al. (2015) has been focused on multiplexed plant genome editing as well as transcriptional regulation. The authors have presented a summary of the CRISPER Cas9 protein, CRISPR RNA, and the CRISPR RNA being the components of the CRISPR Cas 9 system. The two RNA components have been recently identified to be combined with the single gRNA which might be produced in the U6 or U3 small nuclear RNA promoters. The reagent delivery as well as streamline system of the same is enhanced through this process (Li et al., 2021). The authors have identified the hurdles which currently exists regarding plant systems and the limitations which are involved and hence causing issues with the application. The significant molecular expertise in the plant biological system still requires certain efforts to generate the functional expression which will allow the simultaneous editing of the transcriptional regulation. A significant advantage of the CRIPS/Cas 9 system in the genome editing of the plant system is thus analysed (Aman, Mahas & Mahfouz, 2020). The study is focused on development and implementation of the comprehensive molecular toolbox for the multifaceted utilisation of the transcriptional regulation application in plants. The toolbox would act in protocol and with reagents which efficiently and quickly assembles the functional CRISPR/Cas 9 mechanism thus transferring the DNA constructs for the dicots and monocots while utilising the Gateway and Golden Gate cloning methods. The multiplexed gene editing as well as transcriptional repression or activation of the plant endogenous genes has been reported by the authors to report the effectiveness and functionality of the model plants in models such as rice, tobacco, and Arabidopsis.

Harnessing the CRISPR Cas system for programmable transcriptional and post-transcriptional regulation

The details of the evolution of and the mechanism of the CRISPR Cas 9 system has been demonstrated in the present article. The article presents details about the CRISPR Cas 9 module being an RNA guided adaptive immune system, which delivers immunity against the phages and the conjugative plasmids. The molecular mechanisms which are effective in this case and the categorisation of the CRISPR Cas systems have been elaborated upon. The authors have also demonstrated the repercussion of the everlasting race between the phages and the bacteria which is being identified through the CRISPR Cas system. The compositions of the CRISPR Cas 9 system has been demonstrated by the authors and the later directions for the degradation and recognition of the invading DNA (Demirer et al., 2021). The Cas 9 and the ilk proteins which possess unique properties have been presented with numerous options which include the transcription regulation, as well as transcription regulation, the empowering of the researchers, which edit the genes with proper precision and speed.

The benchmarking of the TALE as well as CRISPR  d Cas 9 based transcriptional regulators in various mammalian cells for the manufacture of the synthetic genetic circuits have been demonstrated in details by Lebar and Jerala in their article. The authors have stated that both the technologies have been recently identified to target the DNA segments which are seen rarely in complicated genomes. The sophisticated synthetic circuits which are based on this sytems have been easier to build, however the TALEs have been identified to have several advanatages in this context, The TALE based transcriptional activators have been more effective in this regard than the dCas9 based activators for the induction of transcription. The synthesis circuits of the two main requirements include the orthogonality and efficiency. The authors designed the reporter plasmid to be a single TALE DNA site which was incorporated in the mCitrine fluorescent protein nucleotide sequence specifically as a terminal in frame fusion of the TALE and dCas 9. The technological breakthrough presented by these two mechanisms have not been limited to the genome editing process but also in the building of the designed genetic circuits. The platforms are capable of targeting the rarely occurring segments of DNA which is the primary breakthrough identified in this regard. The mechanism can be utilised to construct genetically engineered logic circuits. The same DNA sequences were targeted by the authors in this case by both tools and the comparison of the advantages in the construction of the engineered logical circuits were determined (Escalona?Noguero et al., 2021). The optimal targeting of the strands for the repression as well as activation of the dCas 9 designed transcription had been functionally complete NOR gates. The CRISPR/Cas 9 system was easy to be constructed however the TALE based activators were stronger and difficult to construct. Nevertheless, the TALE based regulation worked better especially in case of the construction of the layered circuits (Raschmanová et al., 2018).  

Conclusion and future directions

The articles hence discussed in this literature review have provided valuable insight into the specific issues and developments that scientists have been involved in. Future directions in this regard will include the better involvement and designing of tools or platforms which would enable the CRISPR Cas system to be incorporated in the plant genome. The applicability of the foreign DNA as well as the RNA guided modifications through this system holds much potential. The researches should thus be based on developing the CRISPR Cas systems which would be effective in human models as well as plant models. Future researchers should also be in the direction which ensures that the localisation of the Cas 9 genes should be effective and applicable in various domains (Wilson, O Brien & Bauer, 2018). The future research should thus be focused on improving the accuracy of the predictive models which will incorporate the additional features. The current methods are focused in specific sequences of the target site and hence, the future directions would be to ensure the applicability in various sequences, if possible, simultaneously. The incorporation of the various chromatin environments would most probably improve the off-target predictions and hence the susceptibility of the chromatin accessibility would be improved. The inclusion of variant information would also be a step in the future considering the application of the CRISPR technology would be specific and more accurate. The prediction of the possible outcomes would also ensure that the applicability of the CRISPR technology is improved. The prediction of the mutational outcomes of the CRISPR Cas genomic editing would enable the researchers to implement precise edits without involving knock-ins. The improvement of accuracy would thus ensure that the base editing without cleavage would be an attainable goal. The transcription modification system can thus be improved through the CRISPR Cas 9 system.  

CRISPR effectors for transcriptional regulation in human pluripotent stem cells

References

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Andriy Didovyk, Bart?omiej Borek, Lev Tsimring, Jeff Hasty,. Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications, Current Opinion in Biotechnology, Volume 40, 2016, Pages 177-184, ISSN 0958-1669,

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Choi, P. S., & Meyerson, M. (2014). Targeted genomic rearrangements using CRISPR/Cas technology. Nature communications, 5(1), 1-6.https://doi.org/10.1038/ncomms4728

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