Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems

Adams, Sally; Pathak, Prachi; Shao, Hongxia; Lok, James B.; Pires-daSilva, André

doi:10.1038/s41598-018-37036-1

Cited by 53 publications

(43 citation statements)

References 72 publications

(69 reference statements)

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“…In addition, lipofectamine was added to the microinjection mix to improve delivery of the CRISPR components across cell membranes. The Rol phenotype was then observed in a subset of the F 1 progeny of the microinjected females and a subset of the microinjected iL3s (Adams et al, 2019). These results demonstrate that mutant phenotypes can be induced directly in infective larvae through the inclusion of lipofectamine in the microinjection mix, and raise the possibility that a similar approach could be applied to other mammalian-parasitic nematodes such as hookworms that lack a free-living adult stage.…”

Section: Crispr/cas9-mediated Targeted Mutagenesis In Parasitic Nematmentioning

confidence: 73%

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Recent advances in functional genomics for parasitic nematodes of mammals

Castelletto

Gang

Hallem

2020

Journal of Experimental Biology

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Human-parasitic nematodes infect over a quarter of the world's population and are a major cause of morbidity in low-resource settings. Currently available treatments have not been sufficient to eliminate infections in endemic areas, and drug resistance is an increasing concern, making new treatment options a priority. The development of new treatments requires an improved understanding of the basic biology of these nematodes. Specifically, a better understanding of parasitic nematode development, reproduction and behavior may yield novel drug targets or new opportunities for intervention such as repellents or traps. Until recently, our ability to study parasitic nematode biology was limited because few tools were available for their genetic manipulation. This is now changing as a result of recent advances in the large-scale sequencing of nematode genomes and the development of new techniques for their genetic manipulation. Notably, skin-penetrating gastrointestinal nematodes in the genus Strongyloides are now amenable to transgenesis, RNAi and CRISPR/Cas9-mediated targeted mutagenesis, positioning the Strongyloides species as model parasitic nematode systems. A number of other mammalian-parasitic nematodes, including the giant roundworm Ascaris suum and the tissue-dwelling filarial nematode Brugia malayi, are also now amenable to transgenesis and/or RNAi in some contexts. Using these tools, recent studies of Strongyloides species have already provided insight into the molecular pathways that control the developmental decision to form infective larvae and that drive the host-seeking behaviors of infective larvae. Ultimately, a mechanistic understanding of these processes could lead to the development of new avenues for nematode control.

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Section: Crispr/cas9-mediated Targeted Mutagenesis In Parasitic Nematmentioning

confidence: 73%

“…Another recent study used CRISPR to target the S. stercoralis homolog of the C. elegans collagen gene rol-6 (Adams et al, 2019). The CRISPR components were introduced by microinjection into either free-living females or iL3s.…”

Section: Crispr/cas9-mediated Targeted Mutagenesis In Parasitic Nematmentioning

confidence: 99%

Recent advances in functional genomics for parasitic nematodes of mammals

Castelletto

Gang

Hallem

2020

Journal of Experimental Biology

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“…Toward this, we have identified the orthologs of 16 main sex determination genes, including tra-1/2 , gld-1 , her-1 and fem-1/2 (Table S5), which are clear candidates to investigate the sex determination mechanisms in A. rhodensis . In parallel, we are developing reverse genetic and functional genomic technologies for these species, and these promise routes to rapid validation of hypotheses of gene function (Adams et al 2019). The A. rhodensis sex determination system may integrate genetic, maternal, environmental and random components, and this nexus of interacting components will also become amenable to manipulation and dissection.…”

Section: Discussionmentioning

confidence: 99%

“…The RNAi mixture was produced by combining 5 µl of Arh-g5747 siRNA (approximately 100 ng), 4 ul of M9 buffer (Stiernagle 2006) and 1 µl (10% v/v) LipofectamineRNAiMax reagent (Invitrogen) and incubating at 25° for 20 min. Previously, we have shown that inclusion of the transfection reagent Lipofectamine dramatically improves RNAi efficiency in A. rhodensis (Adams et al 2019). For control injections Arh-g5747 siRNA was omitted from the mixture and replaced with additional M9 buffer.…”

Section: Methodsmentioning

confidence: 99%

Chromosome-Wide Evolution and Sex Determination in the Three-Sexed Nematode Auanema rhodensis

Tandonnet

Koutsovoulos

Adams

et al. 2019

G3 Genes|Genomes|Genetics

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Trioecy, a mating system in which males, females and hermaphrodites co-exist, is a useful system to investigate the origin and maintenance of alternative mating strategies. In the trioecious nematode Auanema rhodensis , males have one X chromosome (XO), whereas females and hermaphrodites have two (XX). The female vs. hermaphrodite sex determination mechanisms have remained elusive. In this study, RNA-seq analyses show a 20% difference between the L2 hermaphrodite and female gene expression profiles. RNAi experiments targeting the DM ( doublesex / mab-3 ) domain transcription factor dmd-10/11 suggest that the hermaphrodite sexual fate requires the upregulation of this gene. The genetic linkage map (GLM) shows that there is chromosome-wide heterozygosity for the X chromosome in F2 hermaphrodite-derived lines originated from crosses between two parental inbred strains. These results confirm the lack of recombination of the X chromosome in hermaphrodites, as previously reported. We also describe conserved chromosome elements (Nigon elements), which have been mostly maintained throughout the evolution of Rhabditina nematodes. The seven-chromosome karyotype of A. rhodensis , instead of the typical six found in other rhabditine species, derives from fusion/rearrangements events involving three Nigon elements. The A. rhodensis X chromosome is the smallest and most polymorphic with the least proportion of conserved genes. This may reflect its atypical mode of father-to-son transmission and its lack of recombination in hermaphrodites and males. In conclusion, this study provides a framework for studying the evolution of chromosomes in rhabditine nematodes, as well as possible mechanisms for the sex determination in a three-sexed species.

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“…In addition to these “hands-off” approaches to gene transfer, it is also possible to transfer CRISPR/Cas9 elements into pre-parasitic larvae of soil-transmitted parasites by microinjection (Adams et al, 2019). Mutations were targeted to the rol-6 ortholog in S. stercoralis by microinjecting a nucleoprotein comprising Cas9 and a target specific gRNA along with lipofectamine into the body cavities of iL3.…”

Section: Future Directions In Transgenesis For Parasitic Nematodesmentioning

confidence: 99%

CRISPR/Cas9 Mutagenesis and Expression of Dominant Mutant Transgenes as Functional Genomic Approaches in Parasitic Nematodes

Lok

2019

Front. Genet.

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DNA transformation of parasitic nematodes enables novel approaches to validating predictions from genomic and transcriptomic studies of these important pathogens. Notably, proof of principle for CRISPR/Cas9 mutagenesis has been achieved in Strongyloides spp., allowing identification of molecules essential to the functions of sensory neurons that mediate behaviors comprising host finding, invasion, and location of predilection sites by parasitic nematodes. Likewise, CRISPR/Cas9 knockout of the developmental regulatory transcription factor Ss-daf-16 has validated its function in regulating morphogenesis of infective third-stage larvae in Strongyloides stercoralis . While encouraging, these studies underscore challenges that remain in achieving straightforward validation of essential intervention targets in parasitic nematodes. Chief among these is the likelihood that knockout of multifunctional regulators like Ss -DAF-16 or its downstream mediator, the nuclear receptor Ss -DAF-12, will produce phenotypes so complex as to defy interpretation and will render affected worms incapable of infecting their hosts, thus preventing establishment of stable mutant lines. Approaches to overcoming these impediments could involve refinements to current CRISPR/Cas9 methods in Strongyloides including regulatable Cas9 expression from integrated transgenes and CRISPR/Cas9 editing to ablate specific functional motifs in regulatory molecules without complete knockout. Another approach would express transgenes encoding regulatory molecules of interest with mutations designed to similarly ablate or degrade specific functional motifs such as the ligand binding domain of Ss -DAF-12 while preserving core functions such as DNA binding. Such mutant transgenes would be expected to exert a dominant interfering effect on their endogenous counterparts. Published reports validate the utility of such dominant-negative approaches in Strongyloides .

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Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems

Cited by 53 publications

References 72 publications

Recent advances in functional genomics for parasitic nematodes of mammals

Recent advances in functional genomics for parasitic nematodes of mammals

Chromosome-Wide Evolution and Sex Determination in the Three-Sexed Nematode Auanema rhodensis

CRISPR/Cas9 Mutagenesis and Expression of Dominant Mutant Transgenes as Functional Genomic Approaches in Parasitic Nematodes

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