https://www.alphaknockout.com Mouse Il22ra2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Il22ra2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Il22ra2 gene (NCBI Reference Sequence: NM_178258 ; Ensembl: ENSMUSG00000039760 ) is located on Mouse chromosome 10. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000036564). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Il22ra2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-353K11 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: Mice homozygous for a null mutation display accelerated tumor formation in a colitis associated colon cancer model. Exon 2 starts from about 8.55% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 2303 bp, and the size of intron 2 for 3'-loxP site insertion: 2092 bp. The size of effective cKO region: ~636 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 5 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Il22ra2 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com 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. 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7136bp) | A(28.71% 2049) | C(21.92% 1564) | T(28.36% 2024) | G(21.01% 1499) 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. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr10 + 19621152 19624151 3000 browser details YourSeq 100 605 725 3000 95.5% chr14 + 23205578 23205707 130 browser details YourSeq 92 610 735 3000 93.4% chr11 - 63326625 63326807 183 browser details YourSeq 91 610 725 3000 90.2% chr2 - 5103541 5103652 112 browser details YourSeq 91 609 724 3000 93.5% chr15 - 60418442 60418604 163 browser details YourSeq 91 613 720 3000 97.0% chr16 + 40469663 40469932 270 browser details YourSeq 90 610 725 3000 97.0% chr15 + 68499876 68500130 255 browser details YourSeq 89 615 724 3000 96.9% chr1 - 3589217 3589326 110 browser details YourSeq 88 610 725 3000 89.7% chr14 - 118000145 118000256 112 browser details YourSeq 86 616 722 3000 93.2% chr17 - 67845930 67846051 122 browser details YourSeq 85 617 722 3000 96.8% chr11 + 53112227 53112372 146 browser details YourSeq 84 610 725 3000 95.9% chr17 - 16017904 16018053 150 browser details YourSeq 83 609 725 3000 92.0% chr1 + 165284716 165284844 129 browser details YourSeq 81 614 724 3000 90.4% chr2 - 80452519 80452637 119 browser details YourSeq 77 610 690 3000 98.8% chr13 - 76926781 76926906 126 browser details YourSeq 76 616 706 3000 96.3% chr14 - 25264168 25264257 90 browser details YourSeq 76 610 710 3000 90.3% chr13 - 114992966 114993061 96 browser details YourSeq 76 610 725 3000 88.8% chr10 - 120964321 120964423 103 browser details YourSeq 76 611 722 3000 88.6% chr11 + 48417919 48418062 144 browser details YourSeq 71 627 724 3000 91.2% chr16 + 26065417 26065512 96 Note: The 3000 bp section upstream 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 3000 1 3000 3000 100.0% chr10 + 19624788 19627787 3000 browser details YourSeq 42 2546 2588 3000 100.0% chr16 - 22475031 22475074 44 browser details YourSeq 42 2546 2587 3000 100.0% chr17 + 43398336 43398377 42 browser details YourSeq 41 2546 2586 3000 100.0% chr13 - 46057342 46057382 41 browser details YourSeq 41 2546 2586 3000 100.0% chr12 - 21557709 21557749 41 browser details YourSeq 41 2546 2586 3000 100.0% chr3 + 50272583 50272623 41 browser details YourSeq 41 2546 2586 3000 100.0% chr13 + 95612750 95612790 41 browser details YourSeq 41 2546 2586 3000 100.0% chr12 + 13213057 13213097 41 browser details YourSeq 40 2546 2585 3000 100.0% chr14 - 106606351 106606390 40 browser details YourSeq 40 2545 2584 3000 100.0% chr12 - 29145202 29145241 40 browser details YourSeq 40 2546 2585 3000 100.0% chr11 - 102003133 102003172 40 browser details YourSeq 39 2545 2584 3000 100.0% chr10 - 92262168 92262211 44 browser details YourSeq 39 2548 2586 3000 100.0% chr18 + 38295946 38295984 39 browser details YourSeq 38 2548 2585 3000 100.0% chr3 + 157643096 157643133 38 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Il22ra2 interleukin 22 receptor, alpha 2 [ Mus musculus (house mouse) ] Gene ID: 237310, updated on 1-Oct-2019 Gene summary Official Symbol Il22ra2 provided by MGI Official Full Name interleukin 22 receptor, alpha 2 provided by MGI Primary source MGI:MGI:2665114 See related Ensembl:ENSMUSG00000039760 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as CRF2X; CRF2-10; CRF2-s1; Il-22bp Expression Low expression observed in reference dataset See more Orthologs human all Genomic context Location: 10; 10 A3 See Il22ra2 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (19622028..19634681) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (19341834..19354487) Chromosome 10 - NC_000076.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Il22ra2 ENSMUSG00000039760 Description interleukin 22 receptor, alpha 2 [Source:MGI Symbol;Acc:MGI:2665114] Gene Synonyms Il-22bp Location Chromosome 10: 19,621,998-19,634,681 forward strand. GRCm38:CM001003.2 About this gene This gene has 1 transcript (splice variant), 137 orthologues, 12 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Il22ra2-201 ENSMUST00000036564.7 2498 230aa ENSMUSP00000042642.7 Protein coding CCDS23717 Q80XF5 TSL:1 GENCODE basic APPRIS P1 32.68 kb Forward strand 19.62Mb 19.63Mb 19.64Mb Genes (Comprehensive set... Il22ra2-201 >protein coding Contigs AC153862.8 > Regulatory Build 19.62Mb 19.63Mb 19.64Mb Reverse strand 32.68 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000036564 12.68 kb Forward strand Il22ra2-201 >protein coding ENSMUSP00000042... Low complexity (Seg) Cleavage site (Sign... Superfamily Fibronectin type III superfamily Pfam Fibronectin type III Interferon/interleukin receptor domain PROSITE profiles Fibronectin type III PANTHER PTHR20859 PTHR20859:SF51 Gene3D Immunoglobulin-like fold CDD Fibronectin type III All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 200 230 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.