https://www.alphaknockout.com

Mouse Nr1i3 Knockout Project (CRISPR/Cas9)

Objective: To create a Nr1i3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Nr1i3 gene (NCBI Reference Sequence: NM_009803.5 ; Ensembl: ENSMUSG00000005677 ) is located on Mouse chromosome 1. 9 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000005820). Exon 2 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit decreased sensitivity to TCPOBOP.

Exon 2 starts from the coding region. Exon 2 covers 12.76% of the coding region. The size of effective KO region: ~137 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 9

Legends Exon of mouse Nr1i3 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 137 bp section of Exon 2 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

Overview of the Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 137 bp section of Exon 2 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(137bp) | A(24.82% 34) | C(22.63% 31) | T(21.17% 29) | G(31.39% 43)

Note: The 137 bp section of Exon 2 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(137bp) | A(25.55% 35) | C(22.63% 31) | T(21.17% 29) | G(30.66% 42)

Note: The 137 bp section of Exon 2 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 137 1 137 137 100.0% chr1 + 171214351 171214487 137 browser details YourSeq 26 109 134 137 100.0% chr2 + 27741786 27741811 26 browser details YourSeq 25 110 134 137 100.0% chr15 + 101270943 101270967 25 browser details YourSeq 22 113 134 137 100.0% chr4 + 48052178 48052199 22

Note: The 137 bp section of Exon 2 is BLAT searched against the genome. No significant similarity is found.

BLAT Search Results (down)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 137 1 137 137 100.0% chr1 + 171214349 171214485 137 browser details YourSeq 26 111 136 137 100.0% chr2 + 27741786 27741811 26 browser details YourSeq 25 112 136 137 100.0% chr15 + 101270943 101270967 25 browser details YourSeq 22 115 136 137 100.0% chr4 + 48052178 48052199 22

Note: The 137 bp section of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Nr1i3 nuclear receptor subfamily 1, group I, member 3 [ Mus musculus (house mouse) ] Gene ID: 12355, updated on 26-Jun-2020

Gene summary

Official Symbol Nr1i3 provided by MGI Official Full Name nuclear receptor subfamily 1, group I, member 3 provided by MGI Primary source MGI:MGI:1346307 See related Ensembl:ENSMUSG00000005677 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as CAR; MB67; Care2; ESTM32; AA209988; AI551208; CAR-beta Expression Biased expression in liver adult (RPKM 52.5), duodenum adult (RPKM 13.0) and 3 other tissuesS ee more Orthologs human all

Genomic context

Location: 1 H3; 1 79.21 cM See Nr1i3 in Genome Data Viewer Exon count: 9

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (171213934..171218845)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (173144101..173148975)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 8 transcripts

Gene: Nr1i3 ENSMUSG00000005677

Description nuclear receptor subfamily 1, group I, member 3 [Source:MGI Symbol;Acc:MGI:1346307] Gene Synonyms CAR, CAR1, Care2, ESTM32, MB67, mCAR Location Chromosome 1: 171,213,970-171,220,701 forward strand. GRCm38:CM000994.2 About this gene This gene has 8 transcripts (splice variants), 160 orthologues, 19 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nr1i3- ENSMUST00000005820.10 1414 358aa ENSMUSP00000005820.4 Protein coding CCDS15480 O35627 TSL:1 201 GENCODE basic APPRIS P3

Nr1i3- ENSMUST00000111328.7 1359 357aa ENSMUSP00000106960.1 Protein coding CCDS56655 Q5FW96 TSL:1 203 GENCODE basic APPRIS ALT2

Nr1i3- ENSMUST00000075469.11 1305 286aa ENSMUSP00000074915.5 Protein coding CCDS56654 O35627 TSL:1 202 GENCODE basic

Nr1i3- ENSMUST00000155126.7 2980 221aa ENSMUSP00000137683.1 Protein coding - Q3UEP1 TSL:2 208 GENCODE basic

Nr1i3- ENSMUST00000133075.7 1446 95aa ENSMUSP00000137852.1 Nonsense mediated - M0QWI8 TSL:1 204 decay

Nr1i3- ENSMUST00000152865.7 2855 No - Retained intron - - TSL:2 207 protein

Nr1i3- ENSMUST00000137298.7 1638 No - Retained intron - - TSL:5 205 protein

Nr1i3- ENSMUST00000149404.1 1272 No - Retained intron - - TSL:5 206 protein

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26.73 kb Forward strand 171.21Mb 171.22Mb 171.23Mb Genes (Comprehensive set... Nr1i3-201 >protein coding Apoa2-204 >protein coding

Nr1i3-202 >protein coding Apoa2-201 >protein coding

Nr1i3-208 >protein coding Apoa2-203 >protein coding

Nr1i3-205 >retained intron Apoa2-202 >protein coding

Nr1i3-204 >nonsense mediated decay

Nr1i3-207 >retained intron

Nr1i3-203 >protein coding

Nr1i3-206 >retained intron

Contigs < AC163497.14 < AC084821.26 Genes < Tomm40l-201protein coding < Fcer1g-201protein coding (Comprehensive set...

< Tomm40l-202protein coding < Fcer1g-203protein coding

< Tomm40l-203protein coding

< Tomm40l-206nonsense mediated decay

< Tomm40l-205protein coding

< Tomm40l-207protein coding

< Tomm40l-208processed transcript

< Tomm40l-204retained intron

Regulatory Build

171.21Mb 171.22Mb 171.23Mb Reverse strand 26.73 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000005820

4.88 kb Forward strand

Nr1i3-201 >protein coding

ENSMUSP00000005... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57716 Nuclear hormone receptor-like domain superfamily

SMART Zinc finger, nuclear hormone receptor-type Nuclear hormone receptor, ligand-binding domain

Prints Nuclear hormone receptor

Thyroid hormone receptor

Zinc finger, nuclear hormone receptor-type Pfam Zinc finger, nuclear hormone receptor-type Nuclear hormone receptor, ligand-binding domain

PROSITE profiles Nuclear hormone receptor, ligand-binding domain

Zinc finger, nuclear hormone receptor-type PROSITE patterns Zinc finger, nuclear hormone receptor-type PANTHER PTHR24082

PTHR24082:SF231 Gene3D Zinc finger, NHR/GATA-type Nuclear hormone receptor-like domain superfamily

CDD cd07156

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant stop retained variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 358

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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