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

Abstract

Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.

Figure 1

Morphological differences between the gonads of the genus Auanema and C. elegans. (A) Schematic diagram illustrating the arrangement of the gonad arms in Auanema (top) and C. elegans (bottom). The distal gonad arm of A. rhodensis (B) and A. freiburgensis (C) is smaller, contains fewer, larger nuclei compared to C. elegans (D). The white arrow highlights the distal arm. Scale bar is 50 µM.

Figure 2

CRISPR-Cas9 mediated gene editing of rol-6 homologues in A. rhodensis, A. freiburgensis and S. stercoralis results in a right-handed roller phenotype. (A) Alignment of the predicted protein sequences of the ROL-6 homologues encoded by Cel-rol-6, Arh-rol-6.1 (MH124552), Afr-rol-6.1 (MH124551), S. stercoralis (SSTP_0000742500) and Ppa-prl-1. All show conservation of the arginine residue (*) which is converted to cysteine in ROL-6 (su1006) in C. elegans and Ppa-PRL-1 (tu92) in P. pacificus. (B) Arrangement of the crRNAs, PAM sites and ssDNA donor fragment used for conversion of the Arh-rol-6.1 gene in A. rhodensis. The black arrows indicate the position of gene-specific regions of the anti-parallel crRNA pairs and the grey arrows their corresponding PAM sites. crRNAs represented by forward arrows bind to the non-coding strand, whilst those shown as reverse arrows bind the coding strand. Used in combination, the crRNA antiparallel pairs should result in Cas9-mediated double-stranded DNA breaks on either side of the targeted region. A similar method was used to target Afr-rol-6.1 and the S. stercoralis locus SSTP_0000742500 (Fig. S6). (C) Track pattern of A. rhodensis adult hermaphrodite 200 min after injection with the gene conversion complexes targeting Arh-rol-6.1, as described in (B). Immediately after injection, the individual leaves a wild-type sinusoidal wave as it moves through the bacterial lawn (black arrow highlights the starting point). The pattern becomes increasingly circular as the nematode’s movement becomes locked in a clockwise twisting motion, characteristic of the roller phenotype. (D) Right-roller phenotype (bottom panel) compared to wild-type movement (top panel) in A. rhodensis, A. freiburgensis (adults and larvae) and S. stercoralis (post free-living L2). Scale bar is 1 mm.

Figure 3

Lipofectamine significantly enhances RNAi against the twitchin homologues, Arh-unc-22 and Afr-unc-22, in A. rhodensis and A. freiburgensis, respectively. (A,B) Microinjection of dsRNA alone (−lipofectamine) produced wild type progeny. In contrast, inclusion of Lipofectamine®RNAiMax reagent (+lipofectamine) in the injection mix (C,D) resulted in thinner, paler, straight progeny, which exhibited uncontrolled muscle spasms. Examples shown are young adults (A–C) or a mixture of young adults and larvae (D). Scale bar is 200 µM.

Figure 4

Microinjection of liposome-DNA complexes facilitates expression of the Peef-1A.1::TurboRFP::tbb-2-3′UTR transgene in A. rhodensis. Expression was primarily limited to late embryogenesis in F1 progeny. Scale bar 50 µM.