Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Summary

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F₀ larvae.

Abstract

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Introduction

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. Two lineages of teleost fish, osteoglossiformes and siluriformes, evolved electroreception in parallel, and five lineages of teleosts (gymnotiformes, mormyrids, and the genera Astroscopus, Malapterurus, and Synodontis) evolved electrogenesis in parallel. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture^1,2,3.

This is perhaps best exemplified by the numero....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University.

1. Selecting sgRNA Targets

NOTE: A protocol is provided for manual design of sgRNAs in step 1.1. This was utilized for scn4aa target selection. An additional protocol is provided to facilitate this process (step 1.2) using the EFISHGENOMICS web portal. It is advised that users select protocol 1.2, which featur.......

Discussion

The phenotypic richness of weakly electric fish, together with a recent proliferation of genomics resources, motivates a strong need for functional genomic tools in the weakly electric fish model. This system is particularly attractive because of the convergent evolution of numerous phenotypic traits in parallel lineages of fish, which are easily kept in the laboratory.

The protocol described here demonstrates the efficacy of the CRISPR/Cas9 technique in lineages of weakly electric fish that e.......

Materials

Name	Company	Catalog Number	Comments
20 mg/mL RNA grade Glycogen	Thermo Scientific	R0551
50 bp DNA ladder	NEB	N3236L
borosilicate glass capillary with filament	Sutter Instrument	BF100-58-10	(O.D. 1.0mm, I.D. 0.58 mm, 10 cm length)
Cas9 protein with NLS; 1 mg/mL	PNA Biology	CP01
Dneasy Blood & Tissue Kit	Qiagen	69506
Eppendorf FemptoJet 4i Microinjector	Fisher Scientific	E5252000021
Eppendorf Microloader Pipette Tips	Fisher Scientific	10289651
Hamilton syringe	Fisher Scientific	14-824-654	referred to as "precision glass syringe" in the protocol
Kimwipe	Fisher Scientific	06-666	referred to as "delicate task wipe" in the protocol
MEGAscript T7 Transcription Kit	Invitrogen	AM1334
NEBuffer 3	NEB	B7003S	used for in vitro cleavage assay
OneTaq DNA kit	NEB	M0480L
Ovaprim	Syndel USA	https://www.syndel.com/ovaprim-ovammmlu010.html	referred to as "spawning agent" in the protocol
Parafilm	Fisher Scientific	S37440	referred to as "thermoplastic" in the protocol
Pipette puller	WPI	SU-P97	sutter brand
QIAquick PCR Purification Kit	Qiagen	28106
Reusable needle- requires customization	Fisher Scientific	7803-02	Customize to 0.7 inches long; point style 4 and angle 25
T4 DNA polymerase	NEB	M0203L	Use with the 10X NEB buffer that is included
Teflon coated tools	bonefolder.com	T-SPATULA4PIECE	referred to as "polytetrafluoroethene" in the protocol