https://www.alphaknockout.com Mouse Il18rap Knockout Project (CRISPR/Cas9) Objective: To create a Il18rap knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Il18rap gene (NCBI Reference Sequence: NM_010553 ; Ensembl: ENSMUSG00000026068 ) is located on Mouse chromosome 1. 13 exons are identified, with the ATG start codon in exon 4 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000027237). Exon 4~7 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: Homozygous null mice exhibit defective IL-18-mediated immune responses such as the inability of splenocytes, T helper 1 cells and neutrophils to produce cytokines in response to IL-18. Exon 4 starts from the coding region. Exon 4~7 covers 39.47% of the coding region. The size of effective KO region: ~7411 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 4 5 6 7 13 Legends Exon of mouse Il18rap Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 4 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 2000 bp section downstream of Exon 7 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(23.85% 477) | C(22.95% 459) | T(31.55% 631) | G(21.65% 433) Note: The 2000 bp section upstream of Exon 4 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(2000bp) | A(27.8% 556) | C(20.45% 409) | T(27.75% 555) | G(24.0% 480) Note: The 2000 bp section downstream of Exon 7 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr1 + 40522314 40524313 2000 browser details YourSeq 44 705 784 2000 94.0% chr13 - 29702656 29702908 253 browser details YourSeq 35 780 822 2000 94.8% chr3 - 51805037 51805095 59 browser details YourSeq 33 776 822 2000 84.7% chr1 + 9193084 9193128 45 browser details YourSeq 26 793 822 2000 93.4% chr17 + 36094391 36094420 30 browser details YourSeq 25 1478 1505 2000 84.7% chr8 - 98142226 98142251 26 Note: The 2000 bp section upstream of Exon 4 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 2000 1 2000 2000 100.0% chr1 + 40531628 40533627 2000 browser details YourSeq 621 865 1783 2000 88.3% chr11 - 83025644 83026552 909 browser details YourSeq 615 869 1780 2000 87.5% chr4 - 35671689 35672593 905 browser details YourSeq 607 869 1783 2000 86.9% chrX - 160245841 160246737 897 browser details YourSeq 603 912 1783 2000 87.8% chr5 - 122629728 122630598 871 browser details YourSeq 592 865 1783 2000 86.5% chr9 - 81134712 81135652 941 browser details YourSeq 581 865 1783 2000 87.3% chr3 - 16468246 16469162 917 browser details YourSeq 575 911 1783 2000 87.1% chr1 + 73505270 73506142 873 browser details YourSeq 574 912 1783 2000 87.1% chr18 - 28868992 28869859 868 browser details YourSeq 571 878 1783 2000 88.4% chr1 + 132294300 132295239 940 browser details YourSeq 570 863 1783 2000 87.6% chr1 - 154965399 154966345 947 browser details YourSeq 569 982 1783 2000 87.7% chr15 - 29128271 29129080 810 browser details YourSeq 567 874 1783 2000 88.1% chr7 + 39971281 39972366 1086 browser details YourSeq 566 865 1783 2000 88.3% chr13 - 67062420 67063334 915 browser details YourSeq 565 945 1783 2000 88.8% chr11 - 52579959 52580829 871 browser details YourSeq 565 908 1783 2000 85.2% chr17 + 29596990 29597835 846 browser details YourSeq 560 946 1783 2000 85.7% chrX - 166896329 166897169 841 browser details YourSeq 554 976 1783 2000 89.3% chr19 + 29861774 29862580 807 browser details YourSeq 551 868 1783 2000 86.1% chr17 - 44748044 44748927 884 browser details YourSeq 551 911 1783 2000 87.0% chr1 + 9610022 9610881 860 Note: The 2000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Il18rap interleukin 18 receptor accessory protein [ Mus musculus (house mouse) ] Gene ID: 16174, updated on 12-Aug-2019 Gene summary Official Symbol Il18rap provided by MGI Official Full Name interleukin 18 receptor accessory protein provided by MGI Primary source MGI:MGI:1338888 See related Ensembl:ENSMUSG00000026068 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 AcPL; IL-18RAcP; IL-1RAcPL; IL-18Rbeta; IL-18R-beta Summary Interleukin-18 (or interferon-gamma inducing factor) is a proinflammatory cytokine that induces cell-mediated immunity Expression following microbial infection. This gene encodes a member of the interleukin-1 receptor family. The encoded protein is an accessory subunit of the receptor for interleukin-18 and mediates signaling through this cytokine. Mice lacking this gene exhibit a defective cell-mediated immune response. [provided by RefSeq, Jan 2014] Orthologs Biased expression in liver E18 (RPKM 1.1), spleen adult (RPKM 0.9) and 13 other tissues See more human all Genomic context Location: 1; 1 B See Il18rap in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (40515362..40551707) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (40572207..40606148) Chromosome 1 - NC_000067.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Il18rap ENSMUSG00000026068 Description interleukin 18 receptor accessory protein [Source:MGI Symbol;Acc:MGI:1338888] Gene Synonyms AcPL accessory protein-like) Location Chromosome 1: 40,515,362-40,551,705 forward strand. GRCm38:CM000994.2 About this gene This gene has 4 transcripts (splice variants), 159 orthologues, 10 paralogues, is a member of 1 Ensembl protein family and is associated with 18 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Il18rap- ENSMUST00000027237.11 4889 614aa ENSMUSP00000027237.5 Protein coding CCDS14912 Q0VBK3 TSL:1 201 Q9Z2B1 GENCODE basic APPRIS P1 Il18rap- ENSMUST00000159724.1 2447 No - Retained - - TSL:2 202 protein intron Il18rap- ENSMUST00000163057.7 1439 No - Retained - - TSL:2 204 protein intron Il18rap- ENSMUST00000160468.1 622 No - Retained - - TSL:3 203 protein intron 56.34 kb Forward strand 40.51Mb 40.52Mb 40.53Mb 40.54Mb 40.55Mb 40.56Mb Genes (Comprehensive set... Il18rap-201 >protein coding Il18rap-204 >retained intron Il18rap-203 >retained intron Il18rap-202 >retained intron Contigs AC167126.6 > < AC138210.10 Regulatory Build 40.51Mb 40.52Mb 40.53Mb 40.54Mb 40.55Mb 40.56Mb Reverse strand 56.34 kb Regulation Legend CTCF Open Chromatin Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000027237 36.34 kb Forward strand Il18rap-201 >protein coding ENSMUSP00000027... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily Toll/interleukin-1 receptor homology (TIR) domain superfamily SMART Immunoglobulin subtype Toll/interleukin-1 receptor homology (TIR) domain Prints Interleukin-1 receptor type I/II PR01537 Pfam Immunoglobulin Toll/interleukin-1 receptor homology (TIR) domain IL-18Ra-like, immunoglobulin domain PROSITE profiles Immunoglobulin-like domain Toll/interleukin-1 receptor homology (TIR) domain PANTHER PTHR11890:SF23 Interleukin-1 receptor family Gene3D Immunoglobulin-like fold Toll/interleukin-1 receptor homology (TIR) domain superfamily CDD cd00096 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 614 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.