CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns

Taning, Clauvis Nji Tizi; Eynde, Benigna Van; Yu, Na; Ma, Sanyuan; Smagghe, Guy

doi:10.1016/j.jinsphys.2017.01.007

Cited by 109 publications

(58 citation statements)

References 114 publications

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“…Because high mortality occurred in first instar nymphs using the RNAi system, the function of AcInR1 and AcInR2 in wing dimorphism needs further verification. Genome editing tools such as CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat/CRISPR associated) [46] have been used in many insects and this tool will be helpful in exploring the distinct role of AcInR1 and AcInR2 in aphid wing dimorphism and the development of apterous aphids. This study focused on the nymph–adult transition and presented an approach for exploring functions of genes in aphids.…”

Section: Discussionmentioning

confidence: 99%

Silencing of Two Insulin Receptor Genes Disrupts Nymph-Adult Transition of Alate Brown Citrus Aphid

Ding

Shang

Zhang

et al. 2017

IJMS

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Insulin receptors play key roles in growth, development, and polymorphism in insects. Here, we report two insulin receptor genes (AcInR1 and AcInR2) from the brown citrus aphid, Aphis (Toxoptera) citricidus. Transcriptional analyses showed that AcInR1 increased during the nymph–adult transition in alate aphids, while AcInR2 had the highest expression level in second instar nymphs. AcInR1 is important in aphid development from fourth instar nymphs to adults as verified by dsRNA feeding mediated RNAi. The silencing of AcInR1 or/and AcInR2 produced a variety of phenotypes including adults with normal wings, malformed wings, under-developed wings, and aphids failing to develop beyond the nymphal stages. Silencing of AcInR1 or AcInR2 alone, and co-silencing of both genes, resulted in 73% or 60%, and 87% of aphids with problems in the transition from nymph to normal adult. The co-silencing of AcInR1 and AcInR2 resulted in 62% dead nymphs, but no mortality occurred by silencing of AcInR1 or AcInR2 alone. Phenotypes of adults in the dsInR1 and dsInR2 were similar. The results demonstrate that AcInR1 and AcInR2 are essential for successful nymph–adult transition in alate aphids and show that RNAi methods may be useful for the management of this pest.

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Section: Discussionmentioning

confidence: 99%

Silencing of Two Insulin Receptor Genes Disrupts Nymph-Adult Transition of Alate Brown Citrus Aphid

Ding

Shang

Zhang

et al. 2017

IJMS

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“…Genome editing, which enables emerging technologies to disrupt the function of target genes, uses manipulatable artificial DNA endonucleases such as meganucleases, zinc‐finger nuclease (ZFNs), transcription activator‐like effector nuclease (TALENs) and most recently the RNA‐guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system clustered regularly interspaced short palindromic repeats (CRISPR) . The CRISPR/Cas9 system has been successfully applied in a number of insect species, and has recently been used to investigate insecticide resistance mechanisms . To date, by using CRISPR/Cas9 coupled with non‐homologous end joining (NHEJ) and/or homology directed repair (HDR) approaches, the following mutation(s) edited in the nAChRα6 subunit (G275E and P146S of D. melanogaster ), chitin synthase 1 ( CHS1 , I1015M/F/L of D. melanogaster ), ryanodine receptors ( RyR , G4946E/V & M4790I of D. melanogaster and Spodoptera exigua ), cytochrome P450 gene ( CYP9M10 , KO in Culex quinquefasciatus ), cadherin ( CAD , KO in Helicoverpa armigera ), P‐glycoprotein ( P‐gp , KO in S. exigua ), ABCA2 (KO & KI in H. armigera ), and tetraspanin ( TSPAN1 , KO & KI in H. armigera ) were confirmed in vivo to be involved in insect susceptibility to chemical insecticides or Bacillus thuringiensis Cry toxins …”

Section: Introductionmentioning

confidence: 99%

Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella

Wang

et al. 2019

Pest Management Science

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BACKGROUND The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides some advantages over other reverse genetic techniques to investigate the causal relationship between insecticide resistance phenotype and candidate gene. Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) α6 subunit strongly associated with spinosyns resistance in insects, including Plutella xylostella. However, reverse genetic verification of the P. xylostella nAChRα6 has not yet been achieved via an in vivo approach. RESULTS Here, we successfully constructed a homozygous strain (Pxα6‐KO) with a 2‐nt deletion mutation of nAChRα6 by CRISPR/Cas9 coupled with non‐homologous end joining approach in P. xylostella. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3–TM4 loop region. When compared to the background strain IPP‐S, the knockout strain Pxα6‐KO exhibited 229‐ and 1462‐fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios <3‐fold) effects on the toxicities of imidacloprid, abamectin, β‐cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2‐nt deletion mutation of nAChRα6 in the Pxα6‐KO strain. CONCLUSION In vivo functional investigation demonstrates the causality of the Pxα6 truncating mutation with high levels of resistance to spinosyns in P. xylostella. Our results suggest the Pxα6‐KO strain underlies an autosomal, recessive mode of inheritance for spinetoram resistance, and reinforces the association of this gene to the mode of action of spinosyns. © 2019 Society of Chemical Industry

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“…Off-target cleavage might cause unwanted mutations or structure variations at regions homologous to the sgRNA, or occasionally in unpredictable regions. For entomology applications, offtarget effects are of particular concern for gene drive, an approach to control invasive species and disease-carrying insect vectors (Taning et al, 2017). Several studies in B. mori conducted preliminary evaluations; however, none of them could detect off-target effects.…”

Section: Off-target Effectsmentioning

confidence: 99%

Genome editing in Bombyx mori: New opportunities for silkworm functional genomics and the sericulture industry

Smagghe

Xia

2018

Insect Science

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In recent years, research in life sciences has been remarkably revolutionized owing to the establishment, development and application of genome editing technologies. Genome editing has not only accelerated fundamental research but has also shown promising applications in agricultural breeding and therapy. In particular, the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology has become an indispensable tool in molecular biology owing to its high efficacy and simplicity. Genome editing tools have also been established in silkworm (Bombyx mori), a model organism of Lepidoptera insects with high economic importance. This has remarkably improved the level and scope of silkworm research and could reveal new mechanisms or targets in basic entomology and pest management studies. In this review, we summarize the progress and potential of genome editing in silkworm and its applications in functional genomic studies for generating novel genetic materials.

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CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns

Cited by 109 publications

References 114 publications

Silencing of Two Insulin Receptor Genes Disrupts Nymph-Adult Transition of Alate Brown Citrus Aphid

Silencing of Two Insulin Receptor Genes Disrupts Nymph-Adult Transition of Alate Brown Citrus Aphid

Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella

Genome editing in Bombyx mori: New opportunities for silkworm functional genomics and the sericulture industry

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