run_metadata: 34918

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34918	SRR32335089	SRX27670017	SRS24074872	SRP563805	PRJNA1223441	A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression	GSE289562	Transcriptome Analysis	We c ompared gene expression in dissected whole eyes from crim1 / zebrafish compared to wildtype contros at 72 hpf. This work will be published as Le et al. 2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia colobomatous with microcornea MACOM an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects including retinal coloboma optic pallor and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency generating zebrafish that were homozygous for a 2 basepair deletion c.339 340delCT p.Leu112Leufs*3 in crim1. Homozygous crim1 / larvae demonstrated smaller eyes and small and misshapen lenses compared to controls but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 / larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1 q subcomponent c1q amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 / zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 / have two replicates.		pubmed:40114969		wildtype whole eyes 72 hpf sample 1	GSM8794302		source name:Whole eye\|tissue:Whole eye\|cell type:All\|genotype:Wildtype\|geo loc name:missing\|collection date:missing	wildtype whole eyes 72 hpf sample 1	The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality quality scores sequence length distribution and overrepresented adapter/primer sequences. post quality control with FastQC data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 / mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 / mutants with control datasets using an in house Python script.	Whole eye		Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al. 2018 We performed ‘bulk’ RNA Seq experiments in dissected eyes from in crossed crim1 / larvae and controls at 72 hpf according to prior methods Krall et al. 2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al. 2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double stranded DNA post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al. 2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.		tissue:Whole eye\|cell type:All\|genotype:Wildtype	GSM8794302	GSM8794302: wildtype whole eyes 72 hpf sample 1; Danio rerio; RNA Seq	GSM8794302 r1	GSM8794302	1	Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al. 2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 / larvae and controls at 72 hpf according to prior methods Krall et al. 2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al. 2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double stranded DNA post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al. 2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.		RNA-Seq	TRANSCRIPTOMIC	cDNA	SINGLE	ILLUMINA	Illumina HiSeq 4000		SRP563805			crim1-ctr-1_S17_L003_R1_001.fastq.gz	fastq	3007758600.0	45572100.0	GSM8794302 r1	0:66	A:783035033;C:680405728;G:668894784;T:875142229;N:280826	66				783035033	680405728	668894784	875142229	280826	SRX27670017	SRS24074872			Cincinnati Children's Hospital Medical Center												B		usable mapping rate	illumina	hiseq_era	unknown	random_priming	unknown	bulk	bulk	bulk		United States	2025-02-13	Larval	Larval	Eye	Sensory System