CRISPR versus GMOs: Public acceptance and valuation

Shew, Aaron M.; Nalley, Lawton Lanier; Snell, Heather; Nayga, Rodolfo M.; Dixon, Bruce L.

doi:10.1016/j.gfs.2018.10.005

Cited by 157 publications

(140 citation statements)

References 41 publications

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“…Our egt knock-out have shown similar levels of improvement as previous studies with other egt knock-out mutants generated by insertion on the egt locus of a selecting marker (10, 21, 39) and consequently considered as GM virus. From an applied point of view, and although certain countries are currently debating about the consideration of CRISPR/Cas9 mutants as GM or not (41, 42), our results provide new tools for the generation of GM-free virus with improved properties and potential use in the field. This methodology could be extended to other viruses and other viral loci for the generation of new viral strains with improved properties.…”

Section: Discussionmentioning

confidence: 93%

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing

Pazmiño-Ibarra

Mengual

Targovnik

et al. 2019

Preprint

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adaptation of this methodology for the generation of viral knocks-in specific regions of the viral 35 genome. Analysis of the generated mutants revealed that the edition efficiency and the type of 36 changes was variable but relatively high. Depending on the targeted gene, the rate of edition 37 ranged from 10% to 40%. This study established the first report revealing the potential of 38 CRISPR/Cas9 for the edition of baculovirus contributing to the engineering of baculovirus as 39 protein expression vector as well as a biological control agent. 40 Keywords: CRISPR/Cas9, baculovirus, AcMNPV, genome editing, knock-out, knock-in 41 42 43 with the virus (3). During their viral biphasic life cycle, baculoviruses produce two distinct virion 49 phenotypes: occlusion derived viruses (ODV) and budded viruses (BV). While ODV are 50 involved in horizontal virus transmission from insect to insect through structures named 51 occlusion bodies (OB) which have the virus embedded within, BV are involved in spread of the 52 infection from cell to cell (4). 53Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the prototypic baculovirus 54 most common used for biotechnological purpose, has a circular genome of about 134 Kb that 55contains 156 predicted open reading frames (ORF) (5). The baculovirus genomes, and specially 56 AcMNPV genome, have been genetically modified in order to enhance their pesticide potency 57 and increase the quality and quantity of the recombinant protein expressed in the system (6, 7). 58These modifications include either the deletion of non-essential genes for virus survival or 59 infectivity and the insertion of foreign genes. For instance, in term of increase the insecticidal 60 activity of baculovirus, the cry1Ab gene from Bacillus thuringensis and neurotoxins from 61 scorpion venom have been incorporated into the baculovirus genome (8, 9). In addition, the egt 62 gene (Ac15), involved in the inhibition of the molting, has been deleted from the virus 63 significantly improving the speed of killing of the virus (10). Moreover, different attempt has 64 been conducted to improve the heterologous protein expression in the baculovirus expression 65 system (BEVS). It has been reported that simultaneous deletion of non-essential genes for the in 66 produce insertions or deletions (indels), or homology-directed repair (HDR) in presence of a 87 donor template DNA with homology to the sequence flanking the DSB (16-18). Recent 88 developments in this technology have made possible to generated precise modifications into a 89 5 wide variety of viral genomes (19). However, the CRISPR/Cas9 system has not been applied for 90 the edition of baculoviral genomes. 91In this study, we developed for first time a CRISPR/Cas9-assisted method to edit AcMNPV 92 genome in multiple ways and with different purposes. We have shown that the delivering of 93 Cas9-sgRNA ribonucleoprotein (RNP) complex through lipofection in insect cells might be 94 efficient to generate gene knock-out and knock-in. To evaluate potential app...

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Section: Discussionmentioning

confidence: 93%

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing

Pazmiño-Ibarra

Mengual

Targovnik

et al. 2019

Preprint

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“…Therefore, all genome editing tools should not be treated in the same manner, but rather evaluated separately. However, the EU courts recently categorized new genome editing methods as subject to a 2001 directive, which thus defines them as GMO [179,180]. On the contrary, in the USA, a step toward application of next generation techniques has been made, with the use of Cas9-edited plants now allowed [181].…”

Section: Discussionmentioning

confidence: 99%

Safety Aspects of Genetically Modified Lactic Acid Bacteria

Plavec

Berlec

2020

Microorganisms

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Lactic acid bacteria (LAB) have a long history of use in the food industry. Some species are part of the normal human microbiota and have beneficial properties for human health. Their long-standing use and considerable biotechnological potential have led to the development of various systems for their engineering. Together with novel approaches such as CRISPR-Cas, the established systems for engineering now allow significant improvements to LAB strains. Nevertheless, genetically modified LAB (GM-LAB) still encounter disapproval and are under extensive regulatory requirements. This review presents data on the prospects for LAB to obtain ‘generally recognized as safe’ (GRAS) status. Genetic modification of LAB is discussed, together with problems that can arise from their engineering, including their dissemination into the environment and the spread of antibiotic resistance markers. Possible solutions that would allow the use of GM-LAB are described, such as biocontainment, alternative selection markers, and use of homologous DNA. The use of GM-LAB as cell factories in closed systems that prevent their environmental release is the least problematic aspect, and this is also discussed.

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“…Our egt knockout has shown similar levels of improvement as previous studies with other egt knockout mutants generated by insertion on the egt locus of a selecting marker (Cory et al, 2004;Georgievska et al, 2010;O'Reilly & Miller, 1991) and consequently considered as GM virus. From an applied point of view, and although certain countries are currently debating about the consideration of CRISPR/Cas9 mutants as GM or not (Gelinsky & Hilbeck, 2018;Shew, Nalley, Snell, Nayga, & Dixon, 2018), our results provide new tools for the generation of GM-free virus with improved properties and potential use in the field. This methodology could be extended to other viruses and other viral loci for the generation of new viral strains with improved properties.…”

Section: Odv-e26mentioning

confidence: 93%

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing

Pazmiño-Ibarra

Mengual-Martí

Targovnik

et al. 2019

Biotech & Bioengineering

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The clustered regularly interspaced short palindromic repeats (CRISPR) system–associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9‐single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock‐ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock‐ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent.

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CRISPR versus GMOs: Public acceptance and valuation

Cited by 157 publications

References 41 publications

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing

Safety Aspects of Genetically Modified Lactic Acid Bacteria

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing

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