Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

Summary

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

Abstract

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Introduction

The spinal cord of most vertebrates is unable to regenerate following injury, leading to permanent disability. Several salamanders, such as the axolotl, are notable exceptions. The axolotl can fully regenerate a structurally identical spinal cord and completely restore spinal cord function. Much of the regenerative capability of the axolotl spinal cord is due to ependymal cells. These cells line the central canal, and unlike those in mammals, axolotl ependymal cells remain as neural stem cells (NSCs) post-embryonic development. After spinal cord injury (e.g., from a tail amputation), these NSCs proliferate to regrow the ependymal tube and differentiate to replace lost....

Protocol

All animal experiments must be carried out in accordance with local and national regulations on animal experimentation and with approval of the relevant institutional review board.

1. Preparing the CAS9-gRNA RNP mix

1. Design and synthesize gRNAs.
   NOTE: Refer to other publications for designing and synthesizing gRNAs, including one exclusively concerning axolotls^15,20,21,

Discussion

The described protocol allows time and space restricted gene knock-out in the NSCs in the axolotl spinal cord. The current protocol allows specific targeting of NSCs at a defined time and location with high penetrance. It avoids potential undesired effects originating from gene knock-out in NSCs in other regions such as the brain that occur when using the Cre-LoxP system. It also avoids developmental effects originating from a persistent knock-out, allowing the study of gene function focused on regeneration. In addition,.......

Materials

Name	Company	Catalog Number	Comments
Agarose	Sigma-Aldrich	A9539
Benzocaine	Sigma-Aldrich	E1501-100G
Benzocaine 0.03 % (wt/vol)	Mix 500 ml of 10× TBS, 500 ml of 400% (wt/vol) Holtfreter’s solution and 30 ml of 10% (wt/vol) benzocaine stock solution. Fill up the volume to 10 L with dH2O. The solution can be stored at room temperature for up to 6 months.
Benzocaine 10 % (wt/vol)	Mix 50 g of benzocaine in 500 ml of 100% (vol/vol) ethanol. The solution can be stored at room temperature for up to 12 months.
Borosilicate glass capillaries 1.2 mm O.D., 0.94 mm I.D.	Stutter Instrument	BF120-94-8
CaCl2·2H2O	Merck	102382
CAS9 buffer, 10x	Mix 200 mM HEPES and 1.5 M KCl in RNase-free water. Adjust pH to 7.5. Filter sterilize, aliquot and store at −20 °C for up to 24 months
CAS9-NLS protein	PNA Bio	CP03
Cell culture dishes, 10cm	Falcon	351029
Dumont #5 - Fine Forceps	Fine Scientific Instruments	11254-20
Electroporator	Nepa Gene	NEPA21
	BEX	Pulse Generator CUY21EDIT II
Fast Green FCF	Sigma-Aldrich	F7252-5G
Fast Green FCF Solution, 5x	Dissolve 12.5 mg of Fast Green FCF powder in 10 mL of 1× PBS.
Flaming/Brown Micropipette Puller	Stutter Instrument	P-97
Holtfreter’s solution 400% (wt/vol)	Dissolve 11.125 g of MgSO4·7H2O, 5.36 g of CaCl2·2H2O, 158.4 g of NaCl and 2.875 g of KCl in 10 L of dH2O. The solution can be stored at room temperature for up to 6 months.
KCl	Merck	104936
MgSO4·7H2O	Merck	105886
Microloader pipette tips	Eppendorf	5242956003
Micromanipulator	Narishige	MN-153
NaCl	Merck	106404
Pneumatic PicoPump	World Precision Instruments	SYS-PV830
Ring Forceps	Fine Scientific Instruments	11103-09
Stereomicroscope	Olympus	SZX10
Tris base	Sigma-Aldrich	T6066
Tris-buffered saline, 10x	Dissolve 24.2 g of Tris base and 90 g of NaCl in 990 ml of dH2O. Adjust pH to 8.0 by adding 10 ml of 37% (vol/vol) HCl. The solution can be stored at room temperature for up to 6 months.
Tweezers w/Variable Gap 2 Round Platinum Plate Electrode, 10mm diameter	Nepa Gene	CUY650P10