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

Abstract

3Nematodes belong to one of the most diverse animal phyla. However, functional genomic 1 4 studies in nematodes, other than in a few species, have often been limited in their reliability 1 5 and success. Here we report that by combining liposome-based technology with 1 6 microinjection, we were able to establish a wide range of genomic techniques in the newly 1 7 described nematode genus Auanema. The method also allowed heritable changes in dauer 1 8 larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As 1 9 3 1 the door to mechanistic studies in non-model systems. This allows us to ask questions that 3 2 cannot be addressed with well-established genetic model organisms 1,2 . 3 3 3 4

Chromosome-wide evolution and sex determination in a nematode with three sexes

“…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 41 [16]. 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.…”

“…Free-living trioecious nematodes, such as Auanema rhodensis and Auanema freiburgensis [7,15] are much more experimentally and genetically tractable [16], and may permit delineation of the mechanisms of sex determination and of the processes that underlie the origin and maintenance of alternative mating systems. In Auanema, females and hermaphrodites are karyotypically identical, with two X chromosomes (XX), whereas males have only one X chromosome (XO) [17].…”

“…The RNAi mixture was produced by combining 5 µl of Arh.g5747 siRNA (approximately 100 ng), 4 ul of M9 buffer [22] and 1 µl (10% v/v) Lipofectamine®RNAiMax reagent (Invitrogen) and incubating at 25 °C for 20 min. Previously, we have shown that inclusion of the transfection reagent Lipofectamine dramatically improvesRNAi efficiency in A. rhodensis[16]. For control injections Arh.g5747 siRNA was omitted from the mixture and replaced with additional M9 buffer.For RNAi, young hermaphrodites (day 1 of adulthood) were immobilised on dried 2% agarose (w/v) pads in a small drop of undiluted halocarbon oil 700 (Sigma).…”

Towards genetic modification of plant-parasitic nematodes: delivery of macromolecules to adults and expression of exogenous mRNA in second stage juveniles

“…We explored the use of liposomes to facilitate delivery of macromolecules. Liposomes are vesicular lipid bilayers that have long been used to deliver various cargo to cells by transfection (Felgner et al 1987), and more recently to aid microinjection (Adams et al 2019). We therefore encapsulated FITC-oligo into liposomes (CRISPRMAX lipofectamine) before injection, and injected into the gonad of male M. hapla ( Figure 4C).…”

“…These include the widely studied C. elegans (Frøkjaer-Jensen 2013; Friedland et al 2013), C. remanei (Yin et al 2018), and Pristionchus pacificus (Witte et al 2015), but also relatively recently in other more challenging (animal) parasitic species (e.g. Strongyloides spp., Auanema freiburgensis and A. rhodensis (Lok 2019;Adams et al 2019;Gang et al 2017; O'Halloran 2019)). In this paper we aim to develop some of the foundational biology required to deliver a functional reverse genetics "toolkit" to plant-parasitic nematology.…”