Targeted genome engineering in Caenorhabditis elegans

Abstract

The generation of mutants and transgenes are indispensible for biomedical research. In the nematode Caenorhabditis elegans, a series of methods have been developed to introduce genome modifications, including random mutagenesis by chemical reagents, ionizing radiation and transposon insertion. In addition, foreign DNA can be integrated into the genome through microparticle bombardment approach or by irradiation of animals carrying microinjected extrachromosomal arrays. Recent research has revolutionized the genome engineering technologies by using customized DNA nucleases to manipulate particular genes and genomic sequences. Many streamlined editing strategies are developed to simplify the experimental procedure and minimize the cost. In this review, we will summarize the recent progress of the site-specific genome editing methods in C. elegans, including the Cre/LoxP, FLP/FRT, MosTIC system, zinc-finger nucleases (ZFNs), transcriptional activator-like nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease. Particularly, the recent studies of CRISPR/Cas9-mediated genome editing method in C. elegans will be emphatically discussed.

Recent advances in functional genomics for parasitic nematodes of mammals

“…In particular, a number of approaches have now been used in C. elegans to study gene function in a temporally and spatially regulated manner. These include bipartite systems for the spatiotemporal control of transgene expression, such as the cGal4-UAS, Q-, FLP/FRT and Cre/Lox systems (Davis et al, 2008;Flavell et al, 2013;Hoier et al, 2000;Monsalve et al, 2019;Voutev and Hubbard, 2008;Wang et al, 2017); the split cGal4 system for more precise spatial or spatiotemporal control of transgene expression, including in cases where promoters that drive cell-specific expression are not available (Wang et al, 2018); the ZF1/ZIF and auxin inducible degradation (AID) systems for conditional protein degradation (Armenti et al, 2014;Zhang et al, 2015); and Cre/Lox-based systems for conditional gene disruptions (Chen et al, 2016;Kage-Nakadai et al, 2014). The adaptation of these functional genomic tools to parasitic nematodes would greatly facilitate interrogation of parasite-specific genes and behaviors, with the ultimate goal of developing novel defenses against these pathogens.…”

The CARD-CC/Bcl10/paracaspase signaling complex is functionally conserved since the last common ancestor of Planulozoa

“…Recent advances in cnidarian cell culture might enable such functional analysis by transient overexpression (Rabinowitz et al 2016). Also CRISPR/Cas9 is now a viable option in many invertebrate model systems for generation of knock-out or knock-in mutant animals (Ikmi et al 2014;Chen et al 2016). The sea anemone cnidarian model organisms N. vectensis and Aiptasia might, however, not be the best choices because they express a short form of CYLD and do not have one of the typical cnidarian type 1 paracaspase paralogs found in hydra and corals (PCASP-t1C, Figure 1C, S1).…”

The Caenorhabditis elegans Transgenic Toolbox

“…Next, we describe the use of Mos1 transposons for random and targeted single-copy transgene insertions. We proceed to give an overview of current CRISPR/Cas9 techniques for editing endogenous genes but refer readers interested in more detail to a recent, comprehensive WormBook chapter (Dickinson and Goldstein 2016) and several recent reviews (Chen et al 2016;Farboud 2017). We then present methods for conditional gene deletion using FLP and CRE recombinases, as well as several newly developed conditional protein degradation methods that utilize degron tags.…”