Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Summary

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

Abstract

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Introduction

Microinjection is a common laboratory technique used to deliver a small amount of a substance such as DNA, RNA, protein, and other macromolecules into cells or embryos through a glass capillary¹. Microinjection is performed using special equipment setup including a microinjector, micromanipulator, and a microscope². The technique has been used by researchers to genetically modify many organisms through the generation of transgenics, gene knockouts, and gene therapy, with the aim of understanding the dynamics of intracellular components^3,4,

Protocol

This experiment was conducted at the Fish Genetics Research Unit, E. W. Shell Fisheries Research Center, Auburn University, AL. All experimental protocols used in this experiment were approved by the Auburn University Institutional Animal Care and Use Committee (AU-IACUC) before the experiment was initiated. A list of the equipment and supplies used in this experiment can be found in the Materials Table. The following are the steps and procedures for preparation and microinjection of channel catfish one-cell embryos as illustrated in Figure 1.

1. Brood Stock Selection and Spawning

1. Choose healthy cha....

Results

To demonstrate the efficiency of the microinjection protocol, gRNAs designed to target the channel catfish toll/interleukin 1 receptor domain-containing adapter molecule (TICAM1) gene and rhamnose binding lectin (RBL) gene were microinjected.

TICAM 1
DNA sequencing of vectors from individual colonies from pooled, cloned PCR products revealed the indel mutations induced in TICAM 1 gene. The mutation rate was .......

Discussion

A detailed protocol for microinjection of channel catfish embryos to achieve gene knockout was presented. Injection of CRISPR/Cas9 protein in the yolk is much simpler, saves time, and does not require extensive training when compared to microinjecting the blastodisc of the one-cell embryo, which is the common technique for most gene transfer and gene editing with fish^30,42. The current protocol proved to be successful in inducing indels in channel catfish TICAM 1.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reproboost implant	Center of Marine Biotechnology		Luteinizing hormone releasing hormone analog (LHRHa) for induction of ovulation in channel catfish females
TRICAINE-S	Western Chemical. Inc.		For sedation of brood stock fish during hormone injection and egg stripping.
Phenol red	Sigma-Aldrich	P0290	0.5%, sterile filtered
Stereo microscope	Olympus	213709	For visualizing the eggs during microinjection
Microinjector	ASI-Applied Scientific Instrumentation	Model MPPI-3	For the delivery of the injection material into the embryos
Micromanipulator	ASI-Applied Scientific Instrumentation	Model MM33	For holding and controlling the movement of the injection needle.
Eppendorf Microloader	Eppendorf	5242956.003	For loading injection solution into microinjection needles.
Vertical needle puller	David Kopf Instruments	Model 720	For pulling microinjection needles
Cas9 protein	PNA Bio Inc.	CP01	Recombinant Cas9 protein from Streptococcus pyrogenes.
Expand High FidelityPLUS PCR System	Roche Diagnostics, USA		For PCR and amplification of DNA templates to be used in gRNA preparation
Borosilicate glass capillaries	Fisher Scientific		1 mm outer diameter (OD), for making microinjection needles.
Petri dish	VWR	25384-302	For holding the embryos during the microinjection.
Crisco	The J.M. Smucker Company		Vegetable shortening for coating spawning pans and petri dishes.
Holtfreter`s solution	Home Made		59 mM NaCl, 0.67 mM KCl, 2.4 mM NaHCO3, 0.76 mM CaCl2, 1.67 mM MgSO4 to incubate the microinjected embryos till hatch.
Doxycycline hyclate USP (monohydrate)	Letco Medical	690904	Antibiotic added to Holtfreter's solution to 10 ppm to prevent bacterial infections.