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      Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives

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          Abstract

          The origin of the order Hemiptera can be traced to the late Permian Period more than 230 MYA, well before the origin of flowering plants 100 MY later in during the Cretaceous period. Hemipteran species consume their liquid diets using a sucking proboscis; for phytophagous hemipterans their mouthparts (stylets) are elegant structures that enable voracious feeding from plant xylem or phloem. This adaptation has resulted in some hemipteran species becoming globally significant pests of agriculture resulting in significant annual crop losses. Due to the reliance on chemical insecticides for the control of insect pests in agricultural settings, many hemipteran pests have evolved resistance to insecticides resulting in an urgent need to develop new, species-specific and environmentally friendly methods of pest control. The rapid advances in CRISPR/Cas9 technologies in model insects such as Drosophila melanogaster, Tribolium castaneum, Bombyx mori, and Aedes aegypti has spurred a new round of innovative genetic control strategies in the Diptera and Lepidoptera and an increased interest in assessing genetic control technologies for the Hemiptera. Genetic control approaches in the Hemiptera have, to date, been largely overlooked due to the problems of introducing genetic material into the germline of these insects. The high frequency of CRISPR-mediated mutagenesis in model insect species suggest that, if the delivery problem for Hemiptera could be solved, then gene editing in the Hemiptera might be quickly achieved. Significant advances in CRISPR/Cas9 editing have been realized in nine species of Hemiptera over the past 4 years. Here we review progress in the Hemiptera and discuss the challenges and opportunities for extending contemporary genetic control strategies into species in this agriculturally important insect order r.

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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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            Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9

            CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), Cas9 can be reprogrammed to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently-devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.
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              Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

              Summary: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website. Availability and implementation: Cas-OFFinder free access at http://www.rgenome.net/cas-offinder. Contact: baesau@snu.ac.kr or jskim01@snu.ac.kr
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                Author and article information

                Contributors
                Journal
                Front Bioeng Biotechnol
                Front Bioeng Biotechnol
                Front. Bioeng. Biotechnol.
                Frontiers in Bioengineering and Biotechnology
                Frontiers Media S.A.
                2296-4185
                07 June 2022
                2022
                : 10
                : 900785
                Affiliations
                [1] 1 Department of Entomology , University of California, Riverside , Riverside, CA, United States
                [2] 2 Department of Botany & Plant Sciences , University of California, Riverside , Riverside, CA, United States
                [3] 3 Institute for Integrative Genome Biology , University of California, Riverside , Riverside, CA, United States
                Author notes

                Edited by: Irina Häcker, Justus-Liebig University Giessen, Germany

                Reviewed by: Karl Robinson, University of Queensland, Australia

                Leslie Pick, University of Maryland College Park, United States

                *Correspondence: Peter W. Atkinson, peter.atkinson@ 123456ucr.edu

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

                Article
                900785
                10.3389/fbioe.2022.900785
                9209771
                35747496
                7cd62faf-f7d5-4e2c-b32b-ad98b03a04dc
                Copyright © 2022 Pacheco, Walling and Atkinson.

                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
                : 21 March 2022
                : 09 May 2022
                Funding
                Funded by: California Department of Food and Agriculture , doi 10.13039/100006759;
                Categories
                Bioengineering and Biotechnology
                Review

                hemiptera,crispr/cas9,whiteflies,gene editing,mutations,genetic control

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