PUBLICATION

Transcriptomic and Phenotypic Analysis of CRISPR/Cas9-Mediated gluk2 Knockout in Zebrafish

Authors

Yan, Q., Li, W., Gong, X., Hu, R., Chen, L.

ID

ZDB-PUB-220827-16

Date

2022

Source

Genes 13(8): (Journal)

Registered Authors

Chen, Liangbiao

Keywords

RNA-seq, behavior analysis, cold stress, gluk2, knockout

MeSH Terms

Larva/genetics
CRISPR-Cas Systems
Zebrafish Proteins/genetics
Transcriptome*/genetics
Gene Knockout Techniques
Animals
Zebrafish*/genetics
Period Circadian Proteins

(all 8)

PubMed

36011351 Full text @ Genes (Basel)

Abstract

As a subtype of kainite receptors (KARs), GluK2 plays a role in the perception of cold in the periphery sensory neuron. However, the molecular mechanism for gluk2 on the cold stress in fish has not been reported. In this article, real-time PCR assays showed that gluk2 was highly expressed in the brain and eyes of adult zebrafish. To study the functions of gluk2, gene knockout was carried out using the CRISPR/Cas9 system. According to RNA-seq analysis, we selected the differentially expressed genes (DEGs) that had significant differences in at least three tissues of the liver, gill, intestine, skin, brain, and eyes. Gene Ontology (GO) enrichment analysis revealed that cry1ba, cry2, per1b, per2, hsp70.1, hsp70.2, hsp70l, hsp90aa1.1, hsp90aa1.2, hspb1, trpv1, slc27a1b, park2, ucp3, and METRNL were significantly enriched in the 'Response to temperature stimulus' pathway. Through behavioral phenotyping assay, the gluk2^-/- larval mutant displayed obvious deficiency in cold stress. Furthermore, TUNEL (TdT-mediated dUTP Nick-End Labeling) staining proved that the gill apoptosis of gluk2^-/- mutant was increased approximately 60 times compared with the wild-type after gradient cooling to 8 °C for 15 h. Overall, our data suggested that gluk2 was necessary for cold tolerance in zebrafish.

Genes / Markers

Figures

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Expression

Fish	Conditions	Stage	Anatomy	Assay	Figure
AB	standard conditions	Adult	brain	RTPCR	Fig. 1
AB	standard conditions	Adult	eye	RTPCR	Fig. 1
AB	standard conditions	Adult	gill	RTPCR	Fig. 1
AB	standard conditions	Adult	heart	RTPCR	Fig. 1
AB	standard conditions	Adult	integument	RTPCR	Fig. 1
AB	standard conditions	Adult	intestine	RTPCR	Fig. 1
AB	standard conditions	Adult	kidney	RTPCR	Fig. 1
AB	standard conditions	Adult	liver	RTPCR	Fig. 1
AB	standard conditions	Adult	muscle	RTPCR	Fig. 1
AB	standard conditions	Adult	oocyte	RTPCR	Fig. 1

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
grik2^sou9/sou9 (AB)	standard conditions	Days 7-13	whole organism swimming behavior normal process quality, normal	Fig. 3
grik2^sou9/sou9 (AB)	standard conditions	Adult	brain grik2 expression absent, abnormal	Fig. 1
grik2^sou9/sou9 (AB)	standard conditions	Adult	gill grik2 expression absent, abnormal	Fig. 1
grik2^sou9/sou9 (AB)	standard conditions	Adult	integument grik2 expression absent, abnormal	Fig. 1
grik2^sou9/sou9 (AB)	standard conditions	Adult	intestine grik2 expression absent, abnormal	Fig. 1
grik2^sou9/sou9 (AB)	standard conditions	Adult	liver grik2 expression absent, abnormal	Fig. 1
grik2^sou9/sou9 (AB)	cold shock	Days 7-13	whole organism swimming behavior increased process quality, abnormal	Fig. 3
grik2^sou9/sou9 (AB)	cold exposure	Adult	gill apoptotic process increased process quality, abnormal	Fig. 4
grik2^sou9/sou9 (AB)	cold exposure	Adult	whole organism unbalanced, abnormal	Fig. 4
grik2^sou9/sou9 (AB)	cold exposure	Adult	whole organism viability, abnormal	Fig. 4

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
sou9		Small Deletion	grik2

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
grik2	CRISPR1-grik2	CRISPR

Fish

Fish
AB
grik2^sou9/sou9 (AB)

Antibodies

Orthology

Engineered Foreign Genes

Mapping

Yan et al., 2022 ZDB-PUB-220827-16 PMID:36011351

Transcriptomic and Phenotypic Analysis of CRISPR/Cas9-Mediated gluk2 Knockout in Zebrafish

Yan et al., 2022

ZDB-PUB-220827-16
PMID:36011351