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      CRISPR Cpf1 proteins: structure, function and implications for genome editing

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          Abstract

          CRISPR and CRISPR-associated (Cas) protein, as components of microbial adaptive immune system, allows biologists to edit genomic DNA in a precise and specific way. CRISPR-Cas systems are classified into two main classes and six types. Cpf1 is a putative type V (class II) CRISPR effector, which can be programmed with a CRISPR RNA to bind and cleave complementary DNA targets. Cpf1 has recently emerged as an alternative for Cas9, due to its distinct features such as the ability to target T-rich motifs, no need for trans-activating crRNA, inducing a staggered double-strand break and potential for both RNA processing and DNA nuclease activity. In this review, we attempt to discuss the evolutionary origins, basic architectures, and molecular mechanisms of Cpf1 family proteins, as well as crRNA designing and delivery strategies. We will also describe the novel Cpf1 variants, which have broadened the versatility and feasibility of this system in genome editing, transcription regulation, epigenetic modulation, and base editing. Finally, we will be reviewing the recent studies on utilization of Cpf1as a molecular tool for genome editing.

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          Most cited references100

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          A unique chromatin signature uncovers early developmental enhancers in humans.

          Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment. However, identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term 'poised enhancers', are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis.
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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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              Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

              This protocol from Feng Zhang's lab enables genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system, as sgRNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening.
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                Author and article information

                Contributors
                Fatemesafari27@yahoo.com
                zare.biology@gmail.com
                monica.negahdaripour@yahoo.com
                maziar.bm@gmail.com
                +989177125495 , ghasemiy@sums.ac.ir
                Journal
                Cell Biosci
                Cell Biosci
                Cell & Bioscience
                BioMed Central (London )
                2045-3701
                9 May 2019
                9 May 2019
                2019
                : 9
                : 36
                Affiliations
                [1 ]ISNI 0000 0000 8819 4698, GRID grid.412571.4, Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, , Shiraz University of Medical Sciences, ; Shiraz, Iran
                [2 ]ISNI 0000 0001 0666 1211, GRID grid.411301.6, Department of Basic Sciences, Faculty of Veterinary Medicine, , Ferdowsi University of Mashhad, ; Mashhad, Iran
                [3 ]ISNI 0000 0000 8819 4698, GRID grid.412571.4, Pharmaceutical Sciences Research Center, School of Pharmacy, , Shiraz University of Medical Sciences, ; Shiraz, Iran
                [4 ]ISNI 0000 0001 2164 3847, GRID grid.67105.35, Department of Physiology & Biophysics, School of Medicine, , Case Western Reserve University, ; Ohio, USA
                [5 ]ISNI 0000 0000 8819 4698, GRID grid.412571.4, Department of Pharmaceutical Biotechnology, School of Pharmacy, , Shiraz University of Medical Sciences, ; Shiraz, Iran
                Article
                298
                10.1186/s13578-019-0298-7
                6507119
                31086658
                e1276d7a-08ae-4652-ba31-21c924c5db60
                © The Author(s) 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 13 February 2019
                : 20 April 2019
                Categories
                Review
                Custom metadata
                © The Author(s) 2019

                Cell biology
                crispr/cas9,crispr/cpf1,transcription,epigenetic,gene editing,base editing
                Cell biology
                crispr/cas9, crispr/cpf1, transcription, epigenetic, gene editing, base editing

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