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      CRISPR-Cas12a: Functional overview and applications

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          Abstract

          Prokaryotes have developed an adaptive immune system called Clustered regularly interspaced short palindromic repeats (CRISPR) to combat attacks by foreign mobile genetic elements (MGEs) such as plasmids and phages. In the past decade, the widely characterized CRISPR-Cas9 enzyme has been redesigned to trigger a genome editing revolution. Class II type V CRISPR-Cas12a is a new RNA guided endonuclease that has been recently harnessed as an alternative genome editing tool, which is emerging as a powerful molecular scissor to consider in the genome editing application landscape. In this review, we aim to provide a mechanistic insight into the working mechanism of Cas12a, comparing it with Cas9, and eventually provide an overview of its current applications in genome editing and biotechnology applications.

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          Most cited references35

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          Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.

          Prokaryotes contain short DN repeats known as CRISPR, recognizable by the regular spacing existing between the recurring units. They represent the most widely distributed family of repeats among prokaryotic genomes suggesting a biological function. The origin of the intervening sequences, at present unknown, could provide clues about their biological activities. Here we show that CRISPR spacers derive from preexisting sequences, either chromosomal or within transmissible genetic elements such as bacteriophages and conjugative plasmids. Remarkably, these extrachromosomal elements fail to infect the specific spacer-carrier strain, implying a relationship between CRISPR and immunity against targeted DNA. Bacteriophages and conjugative plasmids are involved in prokaryotic population control, evolution, and pathogenicity. All these biological traits could be influenced by the presence of specific spacers. CRISPR loci can be visualized as mosaics of a repeated unit, separated by sequences at some time present elsewhere in the cell.
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            Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.

            Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.
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              Diversity and evolution of class 2 CRISPR–Cas systems

              Class 2 CRISPR–Cas systems are characterized by effector modules that consist of a single multidomain protein. In this Analysis, using a computational pipeline, the authors discover three novel families of class 2 effectors that correspond to three new CRISPR–Cas subtypes and present a comprehensive census of class 2 systems that are encoded in complete and draft bacterial and archaeal genomes.
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                Author and article information

                Contributors
                Journal
                Biomed J
                Biomed J
                Biomedical Journal
                Chang Gung University
                2319-4170
                2320-2890
                05 February 2020
                February 2020
                05 February 2020
                : 43
                : 1
                : 8-17
                Affiliations
                [1]Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen, Copenhagen, Denmark
                Author notes
                [] Corresponding author. Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen, Blegdamsvej 3-B, Copenhagen 2200, Denmark. guillermo.montoya@ 123456cpr.ku.dk
                Article
                S2319-4170(19)30505-0
                10.1016/j.bj.2019.10.005
                7090318
                32200959
                6105c157-c799-42e9-953c-6d0f9d274cf2
                © 2019 Chang Gung University. Publishing services by Elsevier B.V.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 30 August 2019
                : 21 October 2019
                Categories
                Review Article

                crispr-cas12a,rna guided endonucleases,crrna biogenesis,indiscriminate ssdnase,endonuclease recycling,genome editing

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