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      CRISPR/Cas- and Topical RNAi-Based Technologies for Crop Management and Improvement: Reviewing the Risk Assessment and Challenges Towards a More Sustainable Agriculture

      review-article
      1 , 2 , 1 , 1 , 2 , 2 , 3 , 4 , 1 , 1 , 2 , 5 , 6 , 7 , 2 , 5 , 8 , 3 , 2 , 7 , 1 , * , , 6 , * ,
      Frontiers in Bioengineering and Biotechnology
      Frontiers Media S.A.
      exogenous dsRNA, genome editing, gene silencing, nanotechnology, offtargets, public acceptance, regulatory aspects, toxicity

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          Abstract

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system and RNA interference (RNAi)-based non-transgenic approaches are powerful technologies capable of revolutionizing plant research and breeding. In recent years, the use of these modern technologies has been explored in various sectors of agriculture, introducing or improving important agronomic traits in plant crops, such as increased yield, nutritional quality, abiotic- and, mostly, biotic-stress resistance. However, the limitations of each technique, public perception, and regulatory aspects are hindering its wide adoption for the development of new crop varieties or products. In an attempt to reverse these mishaps, scientists have been researching alternatives to increase the specificity, uptake, and stability of the CRISPR and RNAi system components in the target organism, as well as to reduce the chance of toxicity in nontarget organisms to minimize environmental risk, health problems, and regulatory issues. In this review, we discuss several aspects related to risk assessment, toxicity, and advances in the use of CRISPR/Cas and topical RNAi-based technologies in crop management and breeding. The present study also highlights the advantages and possible drawbacks of each technology, provides a brief overview of how to circumvent the off-target occurrence, the strategies to increase on-target specificity, the harm/benefits of association with nanotechnology, the public perception of the available techniques, worldwide regulatory frameworks regarding topical RNAi and CRISPR technologies, and, lastly, presents successful case studies of biotechnological solutions derived from both technologies, raising potential challenges to reach the market and being social and environmentally safe.

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          Most cited references238

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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            Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.

            The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
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              Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.

              Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
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                Author and article information

                Contributors
                URI : https://loop.frontiersin.org/people/1761813/overview
                URI : https://loop.frontiersin.org/people/1757063/overview
                URI : https://loop.frontiersin.org/people/425946/overview
                URI : https://loop.frontiersin.org/people/440743/overview
                URI : https://loop.frontiersin.org/people/439520/overview
                URI : https://loop.frontiersin.org/people/31336/overview
                Journal
                Front Bioeng Biotechnol
                Front Bioeng Biotechnol
                Front. Bioeng. Biotechnol.
                Frontiers in Bioengineering and Biotechnology
                Frontiers Media S.A.
                2296-4185
                28 June 2022
                2022
                : 10
                : 913728
                Affiliations
                [1] 1 Department of Phytopathology , University of Brasília , Brasília, Brazil
                [2] 2 Embrapa Agroenergy , Brasília, Brazil
                [3] 3 Embrapa Genetic Resources and Biotechnology , Brasília, Brazil
                [4] 4 Embrapa Cassava and Fruits , Cruz Das Almas, Brazil
                [5] 5 IPADS-Balcarce (UEDD INTA-CONICET) , Balcarce, Argentina
                [6] 6 SEMPRE AgTech , Chapecó, Brazil
                [7] 7 Embrapa Soybean , Londrina, Brazil
                [8] 8 USDA-ARS , U.S. Horticultural Research Laboratory , Fort Pierce, FL, United States
                Author notes

                Edited by: Carmen Vicien, University of Buenos Aires, Argentina

                Reviewed by: Gabriela Levitus, ArgenBio, Argentina

                Muhammad Sohaib Shafique, Chinese Academy of Agricultural Sciences (CAAS), China

                *Correspondence: Thaís Ribeiro Santiago, thais.santiago@ 123456unb.br ; Hugo Bruno Correa Molinari, hm@ 123456sempre.agr.br

                This article was submitted to Biosafety and Biosecurity, a section of the journal Frontiers in Bioengineering and Biotechnology

                Article
                913728
                10.3389/fbioe.2022.913728
                9274005
                35837551
                b357611f-d980-47ed-85a5-f113ad9b0def
                Copyright © 2022 Touzdjian Pinheiro Kohlrausch Távora, de Assis dos Santos Diniz, de Moraes Rêgo-Machado, Chagas Freitas, Barbosa Monteiro Arraes, Chumbinho de Andrade, Furtado, Osiro, Lima de Sousa, Cardoso, Márcia Mertz Henning, Abrão de Oliveira Molinari, Feingold, Hunter, Fátima Grossi de Sá, Kobayashi, Lima Nepomuceno, Santiago and Correa Molinari.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 06 April 2022
                : 06 June 2022
                Categories
                Bioengineering and Biotechnology
                Review

                exogenous dsrna,genome editing,gene silencing,nanotechnology,offtargets,public acceptance,regulatory aspects,toxicity

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