https://www.alphaknockout.com Mouse Os9 Knockout Project (CRISPR/Cas9) Objective: To create a Os9 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Os9 gene (NCBI Reference Sequence: NM_001171026 ; Ensembl: ENSMUSG00000040462 ) is located on Mouse chromosome 10. 15 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 15 (Transcript: ENSMUST00000164259). Exon 6~12 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: Exon 6 starts from about 28.77% of the coding region. Exon 6~12 covers 51.39% of the coding region. The size of effective KO region: ~2364 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 6 7 8 9 10 11 12 15 Legends Exon of mouse Os9 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 6 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 852 bp section downstream of Exon 12 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(22.65% 453) | C(25.55% 511) | T(29.45% 589) | G(22.35% 447) Note: The 2000 bp section upstream of Exon 6 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(852bp) | A(21.6% 184) | C(26.17% 223) | T(23.59% 201) | G(28.64% 244) Note: The 852 bp section downstream of Exon 12 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% chr10 - 127100296 127102295 2000 browser details YourSeq 106 647 1645 2000 90.7% chr10 - 61770435 61811473 41039 browser details YourSeq 106 636 791 2000 93.6% chr10 + 99754614 99755099 486 browser details YourSeq 101 649 846 2000 94.2% chr8 - 10136616 10137073 458 browser details YourSeq 96 635 787 2000 88.2% chr10 - 84432180 84432559 380 browser details YourSeq 88 640 789 2000 92.4% chr8 - 127082056 127082365 310 browser details YourSeq 88 635 789 2000 93.2% chr8 - 26775095 26775465 371 browser details YourSeq 88 649 778 2000 91.6% chr1 - 42553326 42553619 294 browser details YourSeq 85 651 777 2000 94.9% chr10 - 116660992 116805650 144659 browser details YourSeq 84 645 792 2000 91.3% chr1 - 186863377 186863754 378 browser details YourSeq 83 649 827 2000 82.4% chr10 - 119509665 119509828 164 browser details YourSeq 82 645 780 2000 94.7% chr8 - 12421690 12422074 385 browser details YourSeq 82 646 792 2000 91.0% chr12 + 106190930 106191331 402 browser details YourSeq 81 646 789 2000 92.8% chr1 + 16342655 16342979 325 browser details YourSeq 79 648 775 2000 94.6% chr8 + 8598693 8599124 432 browser details YourSeq 78 681 813 2000 94.4% chr3 + 146249579 146249804 226 browser details YourSeq 78 645 753 2000 93.7% chr1 + 53623782 53623924 143 browser details YourSeq 77 651 777 2000 91.4% chr4 + 132450354 132450613 260 browser details YourSeq 74 664 778 2000 91.3% chr1 - 165101188 165101347 160 browser details YourSeq 74 649 753 2000 94.1% chr10 + 84053822 84054403 582 Note: The 2000 bp section upstream of Exon 6 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 852 1 852 852 100.0% chr10 - 127097080 127097931 852 browser details YourSeq 45 168 393 852 80.0% chr1 - 126143801 126144012 212 browser details YourSeq 30 47 100 852 75.0% chr1 + 186238848 186238891 44 browser details YourSeq 27 653 680 852 100.0% chr10 - 43037494 43037522 29 browser details YourSeq 27 282 312 852 96.8% chr1 + 183270993 183271036 44 Note: The 852 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Os9 amplified in osteosarcoma [ Mus musculus (house mouse) ] Gene ID: 216440, updated on 12-Aug-2019 Gene summary Official Symbol Os9 provided by MGI Official Full Name amplified in osteosarcoma provided by MGI Primary source MGI:MGI:1924301 See related Ensembl:ENSMUSG00000040462 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 AU022351; 4632413K17Rik Expression Ubiquitous expression in duodenum adult (RPKM 33.8), liver adult (RPKM 33.3) and 28 other tissues See more Orthologs human all Genomic context Location: 10; 10 D3 See Os9 in Genome Data Viewer Exon count: 15 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (127094259..127121160, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (126531315..126558216, complement) Chromosome 10 - NC_000076.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Os9 ENSMUSG00000040462 Description amplified in osteosarcoma [Source:MGI Symbol;Acc:MGI:1924301] Gene Synonyms 4632413K17Rik Location Chromosome 10: 127,095,650-127,121,131 reverse strand. GRCm38:CM001003.2 About this gene This gene has 3 transcripts (splice variants), 174 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Os9- ENSMUST00000164259.8 2627 672aa ENSMUSP00000128914.1 Protein coding CCDS48713 Q8K2C7 TSL:1 202 GENCODE basic APPRIS ALT2 Os9- ENSMUST00000080975.5 2458 617aa ENSMUSP00000079770.4 Protein coding CCDS24229 Q8K2C7 TSL:1 201 GENCODE basic APPRIS P3 Os9- ENSMUST00000218798.1 723 96aa ENSMUSP00000151466.1 Nonsense mediated - A0A1W2P6Z8 CDS 5' 203 decay incomplete TSL:3 45.48 kb Forward strand 127.09Mb 127.10Mb 127.11Mb 127.12Mb 127.13Mb Genes Agap2-203 >protein coding (Comprehensive set... Agap2-202 >protein coding Agap2-201 >protein coding Contigs < AC131760.4 Genes (Comprehensive set... < Os9-201protein coding < Os9-202protein coding < Os9-203nonsense mediated decay Regulatory Build 127.09Mb 127.10Mb 127.11Mb 127.12Mb 127.13Mb Reverse strand 45.48 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000164259 < Os9-202protein coding Reverse strand 25.48 kb ENSMUSP00000128... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Cleavage site (Sign... Superfamily SSF50911 Pfam Protein OS9-like PANTHER PTHR15414:SF0 PTHR15414 Gene3D Mannose-6-phosphate receptor binding domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe insertion missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 672 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.