https://www.alphaknockout.com Mouse Il1rapl1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Il1rapl1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Il1rapl1 gene (NCBI Reference Sequence: NM_001160403 ; Ensembl: ENSMUSG00000052372 ) is located on Mouse chromosome X. 11 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000113966). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Il1rapl1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-458F20 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 knock-out allele exhibit premature giant inhibitory postsynaptic currents and parallel fiber- mediated recruitment of molecular layer interneurons. Exon 3 starts from about 3.98% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 470851 bp, and the size of intron 3 for 3'-loxP site insertion: 114072 bp. The size of effective cKO region: ~780 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 3 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Il1rapl1 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. 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(7280bp) | A(31.91% 2323) | C(17.34% 1262) | T(32.57% 2371) | G(18.19% 1324) 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% chrX - 87419553 87422552 3000 browser details YourSeq 348 2255 2945 3000 83.8% chr15 - 55337270 55337963 694 browser details YourSeq 331 2245 2917 3000 82.5% chrX + 23180263 23180924 662 browser details YourSeq 330 2378 2944 3000 84.9% chr11 + 59623428 59624127 700 browser details YourSeq 311 2251 2944 3000 82.4% chrX - 82965028 82965697 670 browser details YourSeq 311 2284 2945 3000 77.6% chr16 + 88118605 88119244 640 browser details YourSeq 305 2245 2933 3000 82.6% chr17 - 12088718 12089388 671 browser details YourSeq 303 2248 2936 3000 78.1% chrX - 17529702 17530383 682 browser details YourSeq 301 2371 2922 3000 78.8% chrX - 38851231 38851773 543 browser details YourSeq 298 2259 2944 3000 81.3% chr8 + 33276291 33276954 664 browser details YourSeq 297 2248 2942 3000 75.2% chrX - 95407647 95408326 680 browser details YourSeq 295 2245 2955 3000 85.0% chr16 - 21502618 21503578 961 browser details YourSeq 294 2422 2944 3000 82.2% chr6 - 122288760 122289279 520 browser details YourSeq 294 2349 2945 3000 82.9% chr12 - 81535429 81535991 563 browser details YourSeq 292 2286 2851 3000 81.2% chr18 - 81006897 81007459 563 browser details YourSeq 289 2246 2944 3000 79.8% chr4 - 80831109 80831801 693 browser details YourSeq 289 2349 2945 3000 79.6% chr17 + 72248396 72248976 581 browser details YourSeq 285 2349 2932 3000 83.3% chr9 - 52509422 52510000 579 browser details YourSeq 285 2190 2942 3000 83.8% chrX + 92313191 92314258 1068 browser details YourSeq 281 2386 2944 3000 80.7% chr14 - 104159140 104159683 544 Note: The 3000 bp section upstream of Exon 3 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 - 87415773 87418772 3000 browser details YourSeq 914 1466 2991 3000 89.9% chr2 - 11886626 12571595 684970 browser details YourSeq 900 756 2400 3000 87.0% chr1 - 6129960 6598033 468074 browser details YourSeq 583 517 1546 3000 86.9% chr15 - 89600850 89601858 1009 browser details YourSeq 576 517 1603 3000 85.8% chr19 + 11433477 11434785 1309 browser details YourSeq 567 585 1578 3000 87.2% chr16 - 38022040 38023031 992 browser details YourSeq 557 1357 2400 3000 88.4% chr11 + 16212004 16725575 513572 browser details YourSeq 554 551 1531 3000 86.1% chr18 + 32874211 32875192 982 browser details YourSeq 547 1779 2649 3000 86.2% chr8 + 5585319 5586366 1048 browser details YourSeq 544 550 1429 3000 86.4% chr1 - 166220900 166221778 879 browser details YourSeq 528 517 1578 3000 86.1% chr5 + 20594472 20595564 1093 browser details YourSeq 512 517 1496 3000 86.6% chr1 - 16143708 16144714 1007 browser details YourSeq 509 517 1547 3000 87.6% chr8 + 98228163 98229211 1049 browser details YourSeq 506 556 1578 3000 85.9% chr17 - 82358818 82359840 1023 browser details YourSeq 474 1777 2410 3000 88.5% chr8 + 22153308 22153939 632 browser details YourSeq 472 1777 2400 3000 88.1% chrX - 139139267 139139888 622 browser details YourSeq 472 1777 2410 3000 89.3% chrX - 129266386 129267021 636 browser details YourSeq 470 1777 2400 3000 88.5% chr3 - 41147713 41148333 621 browser details YourSeq 469 1777 2400 3000 88.6% chr18 - 63562591 63563213 623 browser details YourSeq 469 1779 2410 3000 88.2% chr18 - 45055957 45259142 203186 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Il1rapl1 interleukin 1 receptor accessory protein-like 1 [ Mus musculus (house mouse) ] Gene ID: 331461, updated on 12-Aug-2019 Gene summary Official Symbol Il1rapl1 provided by MGI Official Full Name interleukin 1 receptor accessory protein-like 1 provided by MGI Primary source MGI:MGI:2687319 See related Ensembl:ENSMUSG00000052372 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 IL1R8; MRX34; OPHN4; IL1RAPL; TIGIRR-2; IL1RAPL-1; C030039N24; 6330532G10Rik Expression Biased expression in frontal lobe adult (RPKM 1.3), cortex adult (RPKM 1.1) and 5 other tissues See more Orthologs human all Genomic context Location: X; X C1 See Il1rapl1 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (86740937..88115948, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (83992581..85360962, complement) Chromosome X - NC_000086.7 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Il1rapl1 ENSMUSG00000052372 Description interleukin 1 receptor accessory protein-like 1 [Source:MGI Symbol;Acc:MGI:2687319] Gene Synonyms 6330532G10Rik, IL1R8, IL1RAPL, MRX34, OPHN4, TIGIRR-2 Location Chromosome X: 86,740,870-88,115,645 reverse strand. GRCm38:CM001013.2 About this gene This gene has 3 transcripts (splice variants), 139 orthologues, 10 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Il1rapl1-203 ENSMUST00000113966.7 9133 696aa ENSMUSP00000109599.1 Protein coding CCDS53128 B1ASU0 TSL:5 GENCODE basic APPRIS P1 Il1rapl1-201 ENSMUST00000078875.7 2091 696aa ENSMUSP00000077915.1 Protein coding CCDS53128 B1ASU0 TSL:5 GENCODE basic APPRIS P1 Il1rapl1-202 ENSMUST00000113964.1 1936 383aa ENSMUSP00000109597.1 Protein coding - Q0VDP7 TSL:1 GENCODE basic 1.39 Mb Forward strand 87.0Mb 87.5Mb 88.0Mb Genes Gm14775-201 >processed pseudogene (Comprehensive set... Contigs AL627409.9 > AL844869.5 > Genes (Comprehensive set... < Il1rapl1-203protein coding < Il1rapl1-201protein coding < Gm24812-201misc RNA < Il1rapl1-202protein coding < Gm8855-201processed pseudogene < Gm14776-201processed pseudogene < Gm14774-201processed pseudogene Regulatory Build 87.0Mb 87.5Mb 88.0Mb Reverse strand 1.39 Mb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding Non-Protein Coding pseudogene RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000113966 < Il1rapl1-203protein coding Reverse strand 1.37 Mb ENSMUSP00000109... Transmembrane heli... MobiDB lite 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 Immunoglobulin subtype 2 Prints PR01537 Pfam IL-18Ra-like, immunoglobulin domain Toll/interleukin-1 receptor homology (TIR) domain Immunoglobulin PROSITE profiles Immunoglobulin-like domain Toll/interleukin-1 receptor homology (TIR) domain PANTHER PTHR11890:SF22 Interleukin-1 receptor family Gene3D Immunoglobulin-like fold Toll/interleukin-1 receptor homology (TIR) domain superfamily CDD cd05896 cd00096 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 696 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.