CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Summary

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Abstract

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken ß-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

Introduction

Brain diseases are one of the most dreadful mortal diseases. They often result from genetic mutations and subsequent dysregulation. To understand molecular mechanisms of brain diseases, ever-lasting efforts to decipher the genomes of human patients have discovered a number of potential causative genes. So far, germline genetically engineered animal models have been utilized for in vivo gain-of-function (GOF) and loss-of-function (LOF) analyses of such candidate genes. Due to the accelerated development of functional validation studies, a more feasible and flexible in vivo gene assay system for studying gene function is desirable.

Protocol

All animal experiments were conducted according to animal welfare regulations and have been approved by the responsible authorities (Regierungspräsidium Karlsruhe, approval numbers: G90/13, G176/13, G32/14, G48/14, and G133/14).

1. Generate pU6-sgRNA-Cbh-Cre Plasmids

1. Design the sgRNA to target a gene-of-interest according to the previously published protocol¹⁴.
   NOTE: In this experiment, two sgRNAs are designed to target the mouse Top2b gene and a non-targeted control sgRNA (Table 1). To knockout gene(s) using the CRISPR technology, sgRNAs need to be designed to target....

Discussion

Using exo utero electroporation, we have previously reported siRNA-based in vivo functional analyses of Atoh1 at an early stage of cerebellar granule cell differentiation⁸. Due to siRNA dilution/degradation and exposure of embryos outside the uterine wall, phenotypic analysis of the electroporated granule cells was limited to embryonic stages. However, the current method enabled analysis of the phenotype of postnatal animals.

Our previous study demonst.......

Materials

Name	Company	Catalog Number	Comments
Alexa 488 Goat anti-Chicken	ThermoFisher	A11039	1:400 dilution
Alexa 568 Donkey anti-Mouse	Life Technologies	A-10037	1:400 dilution
Alexa 594 Donkey anti-Rabbit	ThermoFisher	A21207	1:400 dilution
Alexa 647 Donkey anti-Rabbit	Life Technologies	A31573	1:400 dilution
Alkaline Phosphatase (FastAP)	ThermoFisher	EF0654
Autoclave band	Kisker Biotech	150262
BamHI (HF)	NEB	R3136S
BbsI (FastDigest)	ThermoFisher	FD1014
Cellulose Filter Paper (Whatman)	Sigma-Aldrich	WHA10347525
Cloth	Tork	530378
Confocal laser scanning microscope	Zeiss	LSM800
D-Luciferin	biovision	7903-1
DAPI	Sigma-Aldrich	D9542	1:1000 dilution
Disposable plastic molds (Tissue-Tek Cyromold)	VWR	4566
DMEM Glutamax	ThermoFisher	31966047
Donkey serum	Sigma-Aldrich	D9663
EcoRI (HF)	NEB	R3101S
Electro Square Porator	BTX	ECM830
Endofree Maxi Kit	Qiagen	12362
Ethanol	Merck	107017
Eye ointment (Bepanthen)	Bayer	81552983
Fast Green	Merck	104022
FBS	ThermoFisher	10270-016
Filter (0.22 µm)	Merck	F8148
Fluorescent cell imager (ZOE)	Biorad	1450031
Forceps straight	Fine Science Tools	91150-20
Gauze (X100 ES-pads 8f 10 x 10 cm)	Fisher Scientific	15387311
GFP antibody	Abcam	ab13970	1:1000 dilution
Gibson Assembly Master Mix	NEB	E2611S
Glass Capillary with Filament	Narishige	GD1-2
Heating Pad	ThermoLux	463265 / -67
Image Processing software (ImageJ and Fiji)	NIH	-
Insulin syringe (B. Braun OMNICAN U-100)	Carl Roth	AKP0.1
Isoflurane	Zoetis	TU061219
IVIS Lumina LT Series III Caliper	Perkin Elmer	CLS136331
Kalt Suture Needles	Fine Science Tools	12050-02
KAPA HIFI HOTSTART READY mix	Kapa Biosystems	KK2601
Ki67 antibody	Abcam	ab15580	1:500 dilution
Light Pointer	Photonic	PL3000
Liquid blocker pen	Kisker Biotech	MKP-1
Metamizol	WDT	-
Microgrinder	Narishige	EG-45
Microinjector	Narishige	IM300
Micropipette Puller	Sutter Instrument Co.	P-97
Microscope software ZEN	Zeiss	-
Non-sterile Silk Suture Thread (0.12 mm)	Fine Science Tools	18020-50
O.C.T. Compound (Tissue-Tek)	VWR	4583
p27 antibody	BD bioscience	610241	1:200 dilution
Paraformaldehyde	Roth	335.3
PBS (1x)	Life Technologies	14190169
pCAG-EGxxFP	Addgene	50716
Polyethylenimine	Sigma-Aldrich	408727
pX330 plasmid	Addgene	42230
QIAprep Spin Miniprep Kit	Qiagen	27104
QIAquick Gel Extraction Kit	Qiagen	28704
Quick Ligation Kit	NEB	M2200S
Ring Forceps	Fine Science Tools	11103-09
Slides (SuperFrost)	ThermoFisher	10417002
Software for biostatistics (Prism 7)	GraphPad Software, Inc	-
Spitacid	EcoLab	3003840
Stereomicroscope	Nikon	C-PS
Sucrose	Sigma-Aldrich	S5016
Surgical scissors	Fine Science Tools	91460-11
Surgical scissors with blunt tip	Fine Science Tools	14072-10
Suture (Supramid schwarz DS 16, 1.5 (4/0))	SMI	220340
T4 DNA Ligation Buffer	NEB	B0202S
T4 PNK	NEB	M0201S
Tissue scissors Blunt (11.5 cm)	Fine Science Tools	14072-10
TOP2B antibody	Santa Cruz	sc13059	1:200 dilution
Trypsin (2.5 %)	ThermoFisher	15090046
Tweezers w/5mm Ø disk electrodes Platinum	Xceltis GmbH	CUY650P5
Vaporizer	Drägerwerk AG	GS186