CRISPR-Cas9-based Mutagenesis in the Entomopathogenic Nematode Steinernema hermaphroditum and the Maintenance of Mutant Lines

Summary

This article demonstrates CRISPR-Cas9-mediated genome engineering in Steinernema hermaphroditum, an entomopathogenic (EPN: insect-parasitic) nematode and an emerging genetic model. The described technology is useful for creating mutants, allowing for the elucidation of gene functions in nematode biology that are relevant to mutualistic and parasitic symbiosis.

Abstract

Entomopathogenic nematodes (EPNs) in the genus Steinernema and Heterorhabditis maintain mutualistic interactions with Xenorhabdus and Photorhabdus symbiotic bacteria, respectively. Together, these nematode-bacterium pairs infect and kill insect hosts that are primarily larvae from the orders of Lepidoptera and Coleoptera, forming a tractable tripartite system for dissecting the molecular basis of mutualism and parasitism. A key step towards fully utilizing this model is the development of stable and transgenerational genetic  tools in EPNs. Here, we demonstrate a reliable CRISPR–Cas9 genome editing platform in the emerging model Steinernema hermaphroditum, a species that is readily maintained in vivo and in vitro, and is highly amenable to gonadal microinjection. Importantly, its hermaphroditic reproduction greatly streamlines the generation and maintenance of homozygous mutant lines. We provide a detailed protocol for efficient, targeted gene disruption using microinjection-based delivery of Cas9 ribonucleoprotein complexes. As a proof of concept, we modified the conserved muscle-associated gene unc-22, generating a characteristic twitching phenotype that validates targeted mutagenesis in this system. This CRISPR–Cas9 platform opens the door to stable genetic manipulation in S. hermaphroditum, such as transgene expression, and provides a framework that can be extended to additional EPN species of agricultural and ecological importance.

Introduction

Entomopathogenic nematodes (EPNs) are insect-killing parasites that form species-specific mutualistic partnerships with their bacterial symbionts¹. EPNs consist of two families: Steinermatidae and Heterorhabditidae, which associate with Gram-negative bacteria of Photorhabdus and Xenorhabdus spp, respectively^2,3. During the free-living infective juvenile stage (IJ) of Steinernema nematodes, Xenorhabdus bacteria are housed within an intestinal pocket of the nematode known as the receptacle, and upon penetration of the insect host into the hemocoel, the ....

Protocol

The reagents and the equipment used are listed in the Table of Materials.

1. Nematode preparation prior to microinjection

1. Prepare Nematode Growth Media (NGM) plates seeded with bacteria Xenorhabdus griffiniae and Comamonas aquaticus.
   NOTE: Grow S. hermaphroditum nematodes on their native symbiotic bacterium Xenorhabdus griffiniae before microinjection to maximize nematode growth and recover injected animals on bacterium Comamonas aquaticus to increase post-injection survival¹⁴.
   1. Streak bacter....

Results

Two CRISPR RNAs were designed (identified by the Pam site number - Pam 3 and Pam 5) targeting the S. hermaphroditum homolog of unc-22 at previously published Pam sites¹⁴ (Figure 2A). Each crRNA complexed with tracrRNA to form a single guide RNA (sgRNA) and was incorporated into Cas9 to form the ribonucleoprotein as described in step 3.1. In this protocol, two crRNAs were designed to target the same gene; however, if using the co-CRISPR method develop.......

Discussion

Unconventional genetic model animal systems provide valuable opportunities to study eukaryotic gene function within the ecological and physiological contexts in which those traits naturally occur, for example, in the presence of a parasitic host or an organism’s native microbiome. In entomopathogenic nematodes (EPNs), genetic manipulation has relied on some cases of RNAi-based approaches¹³,³². Although RNAi enables functional  genetic studies in some species, it .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar (80-100 Mesh)	Fisher Scientific	BP26411	Also used in LB Pyruvate plates
Agarose	Fisher Scientific	16500500	Also used in genotyping
Alt-R CRISPR-Cas9 tracrRNA	IDT	1072532
Alt-R S.p. Cas9 Nuclease V3	IDT	1081058
Borosilicate needles	World Precision Instrument	1B120F-4
Calcium chloride (CaCl2)	Sigma Aldrich	C4901
Cholesterol	VWR	433	5 mg/mL in ethanol
Comamonas aquatica (DA1877)
Cover glass for agarose pad	Brain research laboratories	#4860-1D	48 x 60 mm no1 thickness
CRISPRScan			https://www.crisprscan.org/
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	472301	0.5 M
DNA Clean and Concentrator-5	Zymo Research	D4014
FemtoJet 4x Microinjector with Grip head set 4 size 0 needle holder	Eppendorf/Calibre	5253000017
Galleria melonella 5th instar larvae	GrubCo		https://www.grubco.com/index.cfm
Gel imaging system	Azure400	AZI400-01
Gel loading dye	Thermo Fisher Scientific	B72
Gelatin (2%)	Sigma Aldrich	G1393
GelRed	Biotium	#41003
Genotyping primers: DNA oligo, 25 nmol	IDT	N/A
Gibco Bacto Peptone	Fisher Scientific	DF0118-17-0
Gibco Bacto Tryptone	Fisher Scientific	DF0123-17-3	Also used in Dark LB, LB Pyruvate plates
Gibco Bacto Yeast Extract	Fisher Scientific	DF0127-17-9	Also used in Dark LB, LB Pyruvate plates
Grip head set 4 size 1 needle holder	Calibre	EPE-5196083008	(paired with borosilicate needle 1B120F-4)
Halocarbon oil 700	Sigma Aldrich	H8898
Hard platinum wire worm pick for daily maintenance	Tritech Research	PT-9901
Inference of CRISPR edits (ICE)	Synthego	N/A	Software used to analyze Sanger sequences to identify indels
KCl	Sigma Aldrich	P9541
Laser needle puller P-2000	Sutter	P-2000G
Magnesium chloride (MgCl2)	Sigma Aldrich	M8266
Magnesium sulfate (MgSO4)	Sigma Aldrich	M7506	For M9 buffer, Also used in NGM
Microloader tip 2x96ST	Calibre	930001007
Micromanipulator Mounting Adapter for Zeiss Axio Observer and Axiovert 200	Tritech Research	NZ-19-2
Microscope	Tritech	SMT1
Nicotine	Sigma Aldrich	N3876
NP-40	Thermo Fisher Scientific	85124
Nuclease Free Duplex Buffer	IDT	11-01-03-01
OneTaq MasterMix	NEB	M0486
Pipette storage box	Sutter Instruments	BX10
Potassium chloride (KCl)	Sigma Aldrich	793590
Potassium phosphate, dibasic (K2HPO4)	Sigma Aldrich	P3786	KOH for NGM
Potassium phosphate, monobasic (KH2PO4)	Sigma Aldrich	P5655	For M9 buffer, Also used in NGM
Proteinase K	Sigma Aldrich	P6556
Quartz needles	Sutter Instruments	QF100-70-10
S. hermaphroditium genome	NCBI		https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_030435675.1/
sh-unc-22 Alt-R CRISPR-Cas9 crRNAs	IDT	N/A
Sodium chloride (NaCl)	VWR	SS0430	Also used in M9, Dark LB, LB Pyruvate plates
Sodium phosphate, dibasic (Na2HPO4.7H20)	Sigma Aldrich	S9390	For M9 buffer
Sodium pyruvate	Sigma Aldrich	P2256	Also used in LB Pyruvate plates
Soft platinum wire worm pick for microinjection	Surepure Chemetals	6824	Platinum 90%, Iridium 10% Alloy Wire, 0.002 inch diameter x 5 ft long
Standard Needle Holder	Tritech Research	HI-7
Steinernema hermaphroditum
Stereo microscope	Leica	Inveta 3
Trehalose	Cole-Parmer	EW-88195-99	0.08 M
Tris	Fisher Scientific	AM9855G	pH 8.0
Tween 20	Sigma Aldrich	P1379
Vertical Micromanipulator Mounting Adapter	Tritech Research	NR2
Xenorhabdus griffiniae (HGB2511)