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      Dead Cas Systems: Types, Principles, and Applications

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          Abstract

          The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular systems used in research and, increasingly, in medical practice. In particular, Cas proteins devoid of nucleolytic activity (dead Cas proteins; dCas) can be used to deliver functional cargo to programmed sites in the genome. In this review, we describe current CRISPR systems used for developing different dCas-based molecular approaches and summarize their most significant applications. We conclude with comments on the state-of-art in the CRISPR field and future directions.

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          Most cited references116

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          Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

          CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.
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            Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

            Technologies that facilitate the targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. Gene activation by the targeted acetyltransferase is highly specific across the genome. In contrast to conventional dCas9-based activators, the acetyltransferase effectively activates genes from enhancer regions and with individual guide RNAs. The core p300 domain is also portable to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a new robust tool for manipulating gene regulation.
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              Expression profiling reveals off-target gene regulation by RNAi.

              RNA interference is thought to require near-identity between the small interfering RNA (siRNA) and its cognate mRNA. Here, we used gene expression profiling to characterize the specificity of gene silencing by siRNAs in cultured human cells. Transcript profiles revealed siRNA-specific rather than target-specific signatures, including direct silencing of nontargeted genes containing as few as eleven contiguous nucleotides of identity to the siRNA. These results demonstrate that siRNAs may cross-react with targets of limited sequence similarity.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                30 November 2019
                December 2019
                : 20
                : 23
                : 6041
                Affiliations
                [1 ]National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health, Moscow 127994, Russia; seegez@ 123456mail.ru (S.B.); kostyusheva_ap@ 123456mail.ru (A.K.); vladimir.chulanov@ 123456rcvh.ru (V.C.)
                [2 ]Institute of Immunology, Federal Medical Biological Agency, Moscow 115522, Russia
                [3 ]Sechenov First Moscow State Medical University, Moscow 119146, Russia
                [4 ]Central Research Institute of Epidemiology, Moscow 111123, Russia
                Author notes
                [* ]Correspondence: dkostushev@ 123456gmail.com ; Tel.: +7-(926)-085-93-37
                Author information
                https://orcid.org/0000-0001-6303-9293
                Article
                ijms-20-06041
                10.3390/ijms20236041
                6929090
                31801211
                344ba1cb-9241-4867-ae62-82ff48d33a1f
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 31 October 2019
                : 28 November 2019
                Categories
                Review

                Molecular biology
                cas9,dcas,transcription,epigenetics,chromatin,cancer,hereditary diseases,inflammatory diseases,infectious diseases,editing

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