SYNCAS: Efficient CRISPR/Cas9 gene-editing in difficult to transform arthropods

De Rouck, Sander; Mocchetti, Antonio; Dermauw, Wannes; Van Leeuwen, Thomas

doi:10.1016/j.ibmb.2023.104068

Cited by 19 publications

(3 citation statements)

References 69 publications

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“…This is good news for researchers because a homozygous mutant could be obtained in a single step and can be parthenogenetically propagated without the need for further crossing, which significantly facilitates downstream analyses. In some haplodiploid arthropods, males carry a haploid genome, although females are diploid, and thus the similar application of CRISPR system to parental females often produces mutant male progeny carrying a single mutant allele hemizygously [ 34 , 35 ]. However, in these cases, multiple crossings and selections are needed to establish homozygous mutant strains.…”

Section: Discussionmentioning

confidence: 99%

Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR

Kondo,

Tanaka,

Kunieda

2024

PLoS Genet

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Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating tardigrades have long been desired. Despite our prior success in somatic cell gene editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades.

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Section: Discussionmentioning

confidence: 99%

Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR

Kondo,

Tanaka,

Kunieda

2024

PLoS Genet

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“…A premature stop codon in the Obp56e gene, producing a loss-of-function allele, influenced host preference behaviour in this species, while RNAi-mediated knockdown of Obp56e in Drosophila melanogaster resulted in reduced avoidance of Morinda citrifolia (Dworkin & Jones, 2009). We propose TuENaC63 in T. urticae as a candidate for functional validation, which can be eUiciently performed in this species using CRISPR/Cas9 (De Rouck et al, 2024). Detoxification genes are often induced by arthropods upon host shifts, but whether these genes drive the evolution of host adaptation is unclear (Rioja et al, 2017).…”

Section: Molecular Determinants Of Mite Adaptationmentioning

confidence: 96%

Independent genetic mapping experiments identify diverse molecular determinants of host adaptation in a generalist herbivore

Villacis-Perez,

Graeve,

Beer

et al. 2024

Preprint

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Interactions between plants and herbivores promote evolutionary change. Studying the evolution of herbivore mechanisms aimed to cope with diUerent host plant species is a critical intersection between evolutionary biology and sustainable pest management. Generalist herbivores are of particular interest, as hybridization between genetically distinct populations can increase the standing genetic variation and therefore the adaptive potential of the species.Tetranychus urticaeis a generalist arthropod known for its adaptive potential, evidenced in its immense host range and ability to develop metabolic resistance to xenobiotics. However, the molecular underpinnings associated with the potential of host adaptation and the consequences of host adaptation in this and many other pests remain elusive. Here, we use two independent, empirical approaches to identify and map the genetic basis of host plant performance and adaptation in genetically distinct populations ofT. urticae. In the first approach, we subject a genetically diverse mite population to tomato selection and map genomic regions linked to the phenotypic evolution of increased reproductive performance. In the second approach, we map genomic regions responsible for performance on tomato by comparing the genomes of pooled individuals from a F2 backcross between populations with high and low reproductive performance. Both approaches revealed specific and shared genomic regions associated with host plant performance and adaptation and key candidate genes were identified. Our findings highlight the power of spider mite genetic approaches to identify the complex genetic basis of host adaptation in a generalist herbivore.

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“…In some haplodiploid arthropods, males carry a haploid genome, although females are diploid, and thus the similar application of CRISPR system to parental females often produces mutant male progeny carrying a single mutant allele hemizygously [34,35]. However, in these cases, multiple crossings and selections are needed to establish homozygous mutant strains.…”

Section: Plos Geneticsmentioning

confidence: 99%

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SYNCAS: Efficient CRISPR/Cas9 gene-editing in difficult to transform arthropods

Cited by 19 publications

References 69 publications

Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR

Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR

Independent genetic mapping experiments identify diverse molecular determinants of host adaptation in a generalist herbivore

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