Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex

Abstract

The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D. pulex and to advance the understanding of this species. In this study, we developed a genome editing system for D. pulex using CRISPR/Cas9 ribonucleoprotein complexes (Cas9 RNPs). We first assembled a CRISPR single-guide RNA (sgRNA) specific to the Distal-less gene (Dll), which encodes a homeodomain transcription factor essential for distal limb development in invertebrates and vertebrates. Then, we injected Cas9 RNPs into eggs and evaluated its activity in vivo by a T7 endonuclease I assay. Injected embryos showed defective formation of the second antenna and disordered development of appendages, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.

Delivery methods for CRISPR/Cas9 gene editing in crustaceans

“…A significant challenge with microinjecting into embryos is to minimally damage them so that they are still able to hatch after microinjection. We will see below in the work done on Daphnia that sucrose solution was used to balance the osmotic pressure between the embryo and external culture medium (Hiruta et al 2018). More importantly, the developmental stage of embryos is critical for hatching success after microinjection, e.g., within 1 h of ovulation in Daphnia (Nakanishi et al 2014).…”

“…A very similar procedure has been developed for D. pulex, where microinjection was first used for RNAi (Hiruta et al 2013) and then successfully extended to CRISPR/Cas9 gene editing (Hiruta et al 2018). A notable difference for D. pulex is that 60 mM sucrose M4 medium is used for microinjection due to the osmotic pressure specific to the embryo of this species.…”

“…A notable difference for D. pulex is that 60 mM sucrose M4 medium is used for microinjection due to the osmotic pressure specific to the embryo of this species. Additionally, a 2% agar plate with 60 mM sucrose M4 solution is used for hatching the injected embryos (Hiruta et al 2018). Embryos usually hatch out in 2-3 days.…”

“…Microinjection of CRISPR/Cas9 gene editing for Daphnia has been done with both plasmids encoding Cas9 and gRNA, and Cas9 enzyme with separate gRNA (Hiruta et al 2018;Kumagai et al 2017;Nakanishi et al 2014). Nakanishi et al (2014) also performed a comparison of CRISPR/ Cas9 gene knockout efficiency with different concentrations of Cas9 enzyme and sgRNA.…”

“…Nonetheless, for emerging model organisms, the delivery methods are not well developed and often involve significant technical challenges. For crustaceans, which are a major part of aquatic ecosystems and contain many species of economic importance, CRISPR/Cas9 gene editing has been successfully carried out in a few key model species such as the branchiopod Daphnia (Hiruta et al 2018;Nakanishi et al 2014), amphipod Parhyale hawaiensis (Martin et al 2016), and decapod Exopalaemon carinicauda (Gui et al 2016). Here we review the delivery techniques for crustacean gene editing and discuss some of the potential techniques that could be used for delivery in the future.…”