CRISPR/Cas9 mediated gene editing in non-model nematode Panagrolaimus sp. PS1159

Abstract

The phylum Nematoda harbors a huge diversity of species in a broad range of ecosystems and habitats. Nematodes share a largely conserved Bauplan but major differences have been found in early developmental processes. The development of the nematode model organism Caenorhabditis elegans has been studied in great detail for decades. These efforts have provided the community with a large number of protocols and methods. Unfortunately, many of these tools are not easily applicable in non-Caenorhabditis nematodes. In recent years it has become clear that many crucial genes in the C. elegans developmental toolkit are absent in other nematode species. It is thus necessary to study the developmental program of other nematode species in detail to understand evolutionary conservation and novelty in the phylum. Panagrolaimus sp. PS1159 is a non-parasitic nematode exhibiting parthenogenetic reproduction and we are establishing the species to comparatively study evolution, biodiversity, and alternative reproduction and survival strategies. Here, we demonstrate the first successful application of the CRISPR/Cas9 system for genome editing in Panagrolaimus sp. PS1159 and the closely related hermaphroditic species Propanagrolaimus sp. JU765 applying the non-homologous end joining and the homology-directed repair (HDR) mechanisms. Using microinjections and modifying published protocols from C. elegans and P. pacificus we induced mutations in the orthologue of unc-22. This resulted in a visible uncoordinated twitching phenotype. We also compared the HDR efficiency following the delivery of different single-stranded oligodeoxynucleotides (ssODNs). Our work will expand the applicability for a wide range of non-model nematodes from across the tree and facilitate functional analysis into the evolution of parthenogenesis, changes in the developmental program of Nematoda, and cryptobiosis.

CRISPR-Cas9 genome editing inSteinernemaentomopathogenic nematodes

“…To seek for a dominant allele that produces a consistent and visible phenotype as a co-CRISPR marker, I targeted Sh-unc-22, predicted to encode a muscle gene termed Twitchin protein in free-living and parasitic nematodes (Gang et al, 2017; Hellekes et al, 2023; Moerman et al, 1988). In nematodes, UNC-22 is crucial in maintaining muscle contraction cycles.…”

“…The CRISPR-Cas9 system has been successfully adopted in various nematode species, including the classical model C. elegans, the necromenic nematode Pristionchus pacificus, the mammalian parasite Strongyloides stercoralis, and other non-model species, such as Aunema , Oscheius, and Panagrolaimus (Adams et al, 2019; Castelletto & Hallem, 2021; Dockendorff et al, 2022; Hellekes et al, 2023; Hiraga et al, 2021; H.-M. Kim et al, 2022; Lok, 2019). In these successful cases, either plasmid encoded Cas9 and guide RNAs or Cas9 RNP were delivered through microinjection into the gonad of a hermaphrodite or a female nematode.…”

Revisiting Hox gene evolution and Hox cluster linkage across Nematoda

“…Further research regarding nematode Hox gene complements that we have identified in the present study should seek to understand whether and where the colinear expression is retained in the phylum. As more species become amenable to molecular methods 48,51,72 , this can be done with tools like in situ hybridization, single-cell RNA-Seq studies, and CRISPR-guided gene knockouts. In summary, our analyses suggest the potential for new and exciting work on Hox genes and regulation of Hox clusters in a broad sampling of Nematoda.…”

Molecular identification of a peroxidase gene controlling body size in the entomopathogenic nematodeSteinernema hermaphroditum

“…The resulting alleles were consistent with a mixture of NHEJ and MMEJ double-strand break repair (Xue and Greene 2021); we saw no evidence of homology-directed repair using the oligonucleotide template we had included. CRISPR-Cas9 is used extensively in established laboratory research animals and is emerging as a powerful tool in a growing variety of nematodes that have not previously been used extensively in laboratory research (Mendez et al 2022;Cadd et al 2022;Hellekes et al 2023;Dutta et al 2023); it is only with our work and with recent work from Mengyi Cao (personal communication) that CRISPR-Cas9 has been extended to entomopathogenic nematodes. We wish to particularly highlight the potential utility of the skpo-2 locus in performing CRISPR studies using other Steinernema entomopathogens.…”