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Mouse Il13ra2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Il13ra2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Il13ra2 gene (NCBI Reference Sequence: NM_008356 ; Ensembl: ENSMUSG00000031289 ) is located on Mouse chromosome X. 14 exons are identified, with the ATG start codon in exon 6 and the TAA stop codon in exon 14 (Transcript: ENSMUST00000033646). Exon 8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Il13ra2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-114D14 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Null mice display a phenotype consistent with attenuated IL13 responsiveness, including abnormal serum protein concentrations, increased frequency of bone marrow macrophage progenitor cells, and abnormal response of tissue macrophage to LPS.

Exon 8 starts from about 19.93% of the coding region. The knockout of Exon 8 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 1030 bp, and the size of intron 8 for 3'-loxP site insertion: 1304 bp. The size of effective cKO region: ~654 bp. The cKO 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 7 8 9 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Il13ra2 Homology arm cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7154bp) | A(30.46% 2179) | C(17.78% 1272) | T(35.24% 2521) | G(16.52% 1182)

Note: The sequence of homologous arms and cKO region 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 3000 1 3000 3000 100.0% chrX - 147395416 147398415 3000 browser details YourSeq 105 1589 1738 3000 92.7% chr14 + 90710633 90710798 166 browser details YourSeq 86 1608 1735 3000 89.2% chr5 - 52992967 52993086 120 browser details YourSeq 82 1606 1735 3000 86.5% chr12 - 85367963 85368086 124 browser details YourSeq 71 1589 1681 3000 89.8% chr18 + 35034861 35034948 88 browser details YourSeq 69 1647 1745 3000 91.7% chr2 - 145682027 145682125 99 browser details YourSeq 68 1590 1682 3000 85.8% chr13 - 21979963 21980045 83 browser details YourSeq 67 1612 1750 3000 76.9% chr14 + 49732240 49732341 102 browser details YourSeq 65 1642 1733 3000 85.0% chr16 + 62688323 62688407 85 browser details YourSeq 60 1652 1759 3000 89.8% chr15 + 79290866 79290971 106 browser details YourSeq 59 1659 1737 3000 91.8% chr5 + 34737573 34737652 80 browser details YourSeq 59 1652 1738 3000 91.5% chr16 + 61036314 61036401 88 browser details YourSeq 58 1529 1730 3000 91.4% chr12 + 68022278 68022655 378 browser details YourSeq 56 1657 1747 3000 95.2% chr15 - 103213681 103213775 95 browser details YourSeq 53 1658 1735 3000 93.4% chr2 - 165044789 165044875 87 browser details YourSeq 51 1626 1735 3000 78.4% chr10 + 65741629 65741720 92 browser details YourSeq 49 1673 1742 3000 89.1% chr10 + 113961861 113961928 68 browser details YourSeq 43 1666 1738 3000 86.3% chr1 + 155143821 155143891 71 browser details YourSeq 41 1704 1747 3000 97.8% chr10 - 41818966 41819010 45 browser details YourSeq 41 1698 1747 3000 91.9% chr1 + 97830271 97830321 51

Note: The 3000 bp section upstream of Exon 8 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 3000 1 3000 3000 100.0% chrX - 147391762 147394761 3000 browser details YourSeq 148 2741 2918 3000 89.9% chr17 + 48305256 48305423 168 browser details YourSeq 147 2763 2918 3000 97.5% chr9 - 76272641 76272800 160 browser details YourSeq 147 2699 2920 3000 93.0% chr3 - 90972793 90973132 340 browser details YourSeq 147 2762 2918 3000 96.9% chr1 + 69268484 69268640 157 browser details YourSeq 146 2763 2918 3000 97.5% chr18 - 29667966 29668137 172 browser details YourSeq 146 2763 2918 3000 96.8% chrX + 81813463 81813618 156 browser details YourSeq 146 2665 2918 3000 87.4% chr12 + 56175608 56175768 161 browser details YourSeq 145 2757 2918 3000 95.1% chr8 - 64165991 64166162 172 browser details YourSeq 145 2762 2918 3000 96.2% chr2 - 172243174 172243330 157 browser details YourSeq 145 2665 2918 3000 86.8% chr8 + 64418001 64418158 158 browser details YourSeq 145 2760 2918 3000 95.6% chr19 + 18201863 18202021 159 browser details YourSeq 144 2759 2918 3000 95.6% chr9 - 55654046 55654220 175 browser details YourSeq 144 2763 2918 3000 96.2% chr3 - 148781211 148781366 156 browser details YourSeq 144 2759 2918 3000 95.7% chr16 - 71991931 71992095 165 browser details YourSeq 144 2763 2918 3000 96.2% chr1 - 87905932 87906087 156 browser details YourSeq 143 2758 2918 3000 95.0% chr6 - 136019960 136020120 161 browser details YourSeq 143 2751 2918 3000 92.8% chr2 - 18275826 18275993 168 browser details YourSeq 143 2758 2918 3000 93.2% chr12 - 74581209 74581368 160 browser details YourSeq 143 2758 2918 3000 93.2% chr9 + 87601807 87601966 160

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Il13ra2 interleukin 13 receptor, alpha 2 [ Mus musculus (house mouse) ] Gene ID: 16165, updated on 10-Oct-2019

Gene summary

Official Symbol Il13ra2 provided by MGI Official Full Name interleukin 13 receptor, alpha 2 provided by MGI Primary source MGI:MGI:1277954 See related Ensembl:ENSMUSG00000031289 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as CD213a2; IL-13R-alpha-2 Summary This gene encodes a receptor protein that binds to interleukin 13 (IL-13) with very high affinity. The encoded protein acts as Expression a decoy receptor, and does not elicit any signal upon the binding of IL-13. Mice lacking the encoded protein exhibit increased levels of serum immunoglobulins, immune-dependent production of interferon gamma and, increased bone marrow macrophage progenitor frequency. Macrophages lacking the encoded protein release less nitric oxide and IL-12 in response to lipopolysaccharide. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Apr 2015] Orthologs Biased expression in bladder adult (RPKM 1.3), frontal lobe adult (RPKM 0.3) and 2 other tissues See more human all

Genomic context

Location: X F2; X 68.46 cM See Il13ra2 in Genome Data Viewer

Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (147383478..147429192, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (143818021..143838375, complement)

Chromosome X - NC_000086.7

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Transcript information: This gene has 2 transcripts

Gene: Il13ra2 ENSMUSG00000031289

Description interleukin 13 receptor, alpha 2 [Source:MGI Symbol;Acc:MGI:1277954] Gene Synonyms CD213a2 Location Chromosome X: 147,383,476-147,429,192 reverse strand. GRCm38:CM001013.2 About this gene This gene has 2 transcripts (splice variants), 185 orthologues, 28 paralogues, is a member of 1 Ensembl protein family and is associated with 17 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Il13ra2-201 ENSMUST00000033646.8 2281 383aa ENSMUSP00000033646.2 Protein coding CCDS30460 O88786 TSL:1 GENCODE basic APPRIS P1

Il13ra2-202 ENSMUST00000112827.1 1536 383aa ENSMUSP00000108446.1 Protein coding CCDS30460 O88786 TSL:1 GENCODE basic APPRIS P1

65.72 kb Forward strand 147.38Mb 147.39Mb 147.40Mb 147.41Mb 147.42Mb 147.43Mb Contigs AL662932.10 > AL807780.7 > Genes (Comprehensive set... < Il13ra2-201protein coding

< Il13ra2-202protein coding

Regulatory Build

147.38Mb 147.39Mb 147.40Mb 147.41Mb 147.42Mb 147.43Mb Reverse strand 65.72 kb

Regulation Legend Open Chromatin

Gene Legend Protein Coding

Ensembl protein coding

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Transcript: ENSMUST00000033646

< Il13ra2-201protein coding

Reverse strand 45.72 kb

ENSMUSP00000033... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily Fibronectin type III superfamily Pfam Type I cytokine receptor, cytokine-binding domain

PROSITE profiles Fibronectin type III PROSITE patterns Short hematopoietin receptor, family 2, conserved site

PANTHER PTHR23036:SF101

PTHR23036 Gene3D Immunoglobulin-like fold

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

Variant Legend

missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 383

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

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