Gonad Microinjection: A Method of Compound Delivery Directly into the Germline of C. elegans

Abstract

Source: Farboud, B. et al, Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms. J. Vis. Exp. (2018).

This video describes microinjection, a common method of creating transgenic C. elegans strains.  In the example, we will see microinjection used to introduce a ribonucleoprotein (RNP) complex for CRISPR-bases gene editing.

Protocol

The following protocol is an excerpt from Farboud et al, Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms, J. Vis. Exp. (2018). 1. RNP Assembly Design the experiment well in advance, acquiring all the RNA, DNA, and protein components ahead of time. As a first pass, try one of…

Representative Results

Table 1: Positive controls for preliminary genome editing experiments. This table shows the key information needed to perform a first-time genome editing experiment in each of the cells and organisms described in this protocol. Following these parameters is likely to yield a successful result that can be used to test the protocol or as a baseline for comparison once the experimenter is targeting a…

Materials

Reagents/Materials
DNA oligonucleotides	Integrated DNA Technologies	–	IDT will provide custom DNA sequences, including those in Table 1
Guide RNAs	Synthego	–	Synthego will provide high-quality sgRNAs for S. pyogenes Cas9, including custom sgRNAs containing the targeting sequences included in Table 1
Purified Cas9 protein (EnGen Cas9 NLS, S. pyogenes)	New England Biosciences	M0646T	If possible, purifying Cas9 in-house or purchasing from local core facilities is a less expensive option
OP50 Escherichia coli	Caenorhabditis Genetics Center	OP-50	https://cgc.umn.edu/
Nematode Growth Media agar in petri dishes	–	–	See Stiernagle, T (ref. 59)
Standard borosilicate glass capillaries with filament:  4 in (100 mm), 1/0.58 OD/ID	World Precision Instruments	1B100F-4
Single-barrel standard borosilicate glass capillaries: 6 in (152 mm), 2/1.12 OD/ID	World Precision Instruments	1B200-6
Cover glass; 24 × 50 mm	Thermo Fisher Scientific	12-544E
Cover glass; 22 × 22 mm	Thermo Fisher Scientific	12-518-105K
Apex LE agarose	Genesee Scientific	20-102
Halocarbon oil 700	Sigma-Aldrich	H8898-100ML
pCFJ90 plasmid	Addgene	19327
Capillary tubes with filament: 4 in (1.0 mm)	World Precision Instruments	T2100F-4
Petri dishes (100 × 15 mm)	–
9" pasteur pipettes	–
Nuclease-free water	–
Equipment
MZ75 Stereomicroscope	Leica	Out-of-production. Current model is the M80 Stereomicroscope
Axio Vert35 inverted phase contrast fluorescent microscope	Zeiss	Out-of-production. Current model is the Axio VertA.1
Laser-based micropipette puller (for C. elegans protocol)	Sutter Instrument	FG-P2000
Picoliter Microinjector (for C. elegans protocol)	Warner Instruments	PLI-100A
Three-axis Joystick oil hydraulic micromanipulator	Narishige International	MO-202U
Coarse manipulator	Narishige International	MMN-1
Microloader pipette tips	Eppendorf	5242956003
NG-agar