https://www.alphaknockout.com Mouse Opcml Knockout Project (CRISPR/Cas9) Objective: To create a Opcml knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Opcml gene (NCBI Reference Sequence: NM_177906 ; Ensembl: ENSMUSG00000062257 ) is located on Mouse chromosome 9. 8 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000115243). Exon 3 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 3 starts from about 14.54% of the coding region. Exon 3 covers 23.05% of the coding region. The size of effective KO region: ~233 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 3 8 Legends Exon of mouse Opcml Knockout region Page 2 of 9 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 3 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 3 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 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(25.05% 501) | C(22.0% 440) | T(34.25% 685) | G(18.7% 374) Note: The 2000 bp section upstream of Exon 3 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(29.75% 595) | C(19.75% 395) | T(29.9% 598) | G(20.6% 412) Note: The 2000 bp section downstream of Exon 3 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 9 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% chr9 + 28673135 28675134 2000 browser details YourSeq 73 578 687 2000 89.8% chr11 + 75000468 75000574 107 browser details YourSeq 70 1859 1987 2000 78.6% chr10 - 99394374 99394464 91 browser details YourSeq 57 1859 1942 2000 95.5% chr11 + 52744497 52744626 130 browser details YourSeq 57 1859 1947 2000 89.8% chr1 + 189692995 189693082 88 browser details YourSeq 51 583 656 2000 95.0% chr10 - 56798030 56798107 78 browser details YourSeq 51 1876 1958 2000 81.4% chr1 + 180602462 180602538 77 browser details YourSeq 50 1856 1938 2000 91.3% chr1 - 130633620 130633703 84 browser details YourSeq 49 1878 1989 2000 71.7% chr14 - 23065976 23066047 72 browser details YourSeq 49 1856 1942 2000 80.4% chr1 + 179671422 179671499 78 browser details YourSeq 47 1859 1926 2000 81.5% chr1 - 187814946 187815005 60 browser details YourSeq 47 1857 1933 2000 91.3% chr1 - 77360929 77361005 77 browser details YourSeq 40 1856 1927 2000 71.0% chr11 - 110386918 110386973 56 browser details YourSeq 40 1856 1958 2000 68.8% chr13 + 18295641 18295704 64 browser details YourSeq 37 1856 1941 2000 67.5% chr14 - 4477571 4477620 50 browser details YourSeq 37 1877 1927 2000 95.3% chr1 - 59224930 59225001 72 browser details YourSeq 37 1792 1943 2000 95.2% chr3 + 86371230 86371381 152 browser details YourSeq 36 1909 1973 2000 75.0% chr3 - 132505526 132505576 51 browser details YourSeq 36 1916 1978 2000 80.0% chr1 - 165046530 165046588 59 browser details YourSeq 36 1873 1917 2000 82.1% chr1 - 82490384 82490423 40 Note: The 2000 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 2000 1 2000 2000 100.0% chr9 + 28675368 28677367 2000 browser details YourSeq 26 858 885 2000 88.9% chr11 + 38449076 38449102 27 browser details YourSeq 22 158 179 2000 100.0% chr2 + 171763630 171763651 22 browser details YourSeq 21 1190 1210 2000 100.0% chr7 - 124882481 124882501 21 browser details YourSeq 21 1330 1351 2000 100.0% chr5 - 70780785 70780807 23 browser details YourSeq 21 137 157 2000 100.0% chr4 + 30474766 30474786 21 browser details YourSeq 21 90 111 2000 100.0% chr3 + 19042751 19042773 23 browser details YourSeq 21 1619 1640 2000 100.0% chr14 + 57169729 57169751 23 browser details YourSeq 20 139 160 2000 95.5% chr1 - 5979526 5979547 22 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Opcml opioid binding protein/cell adhesion molecule-like [ Mus musculus (house mouse) ] Gene ID: 330908, updated on 15-Aug-2019 Gene summary Official Symbol Opcml provided by MGI Official Full Name opioid binding protein/cell adhesion molecule-like provided by MGI Primary source MGI:MGI:97397 See related Ensembl:ENSMUSG00000062257 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 Gm181; Obcam; AI844366; 3732419F12; C230027C17; 2900075O15Rik; B930023M13Rik Expression Biased expression in frontal lobe adult (RPKM 13.6), cortex adult (RPKM 13.0) and 4 other tissues See more Orthologs human all Genomic context Location: 9 A4; 9 13.73 cM See Opcml in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (27790716..28925410) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (27598854..28732643) Chromosome 9 - NC_000075.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Opcml ENSMUSG00000062257 Description opioid binding protein/cell adhesion molecule-like [Source:MGI Symbol;Acc:MGI:97397] Gene Synonyms 2900075O15Rik, B930023M13Rik, LOC235104, Obcam Location Chromosome 9: 27,790,775-28,925,410 forward strand. GRCm38:CM001002.2 About this gene This gene has 9 transcripts (splice variants), 217 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Opcml-202 ENSMUST00000115243.8 6676 337aa ENSMUSP00000110898.2 Protein coding CCDS22942 Q6DFY2 TSL:1 GENCODE basic Opcml-201 ENSMUST00000073822.5 2378 345aa ENSMUSP00000073493.5 Protein coding - G5E8G3 TSL:5 GENCODE basic APPRIS P1 Opcml-204 ENSMUST00000126673.1 2894 No protein - Retained intron - - TSL:1 Opcml-209 ENSMUST00000214395.1 1771 No protein - Retained intron - - TSL:NA Opcml-206 ENSMUST00000133142.1 2592 No protein - lncRNA - - TSL:1 Opcml-205 ENSMUST00000126845.1 730 No protein - lncRNA - - TSL:3 Opcml-203 ENSMUST00000123779.1 665 No protein - lncRNA - - TSL:1 Opcml-208 ENSMUST00000150098.1 632 No protein - lncRNA - - TSL:3 Opcml-207 ENSMUST00000134002.1 351 No protein - lncRNA - - TSL:3 Page 7 of 9 https://www.alphaknockout.com 1.15 Mb Forward strand 27.8Mb 28.0Mb 28.2Mb 28.4Mb 28.6Mb 28.8Mb Genes (Comprehensive set... Opcml-207 >lncRNA Gm15606-201 >lncRNA Opcml-206 >lncRNA Opcml-205 >lncRNA Opcml-202 >protein coding Opcml-209 >retained intron Gm15606-202 >lncRNA Opcml-201 >protein coding Opcml-203 >lncRNA Gm44316-201 >miRNA Opcml-204 >retained intron Opcml-208 >lncRNA Contigs < AC116575.4 AC102044.7 > < AC157511.2 AC154803.2 > < AC161272.3 AC116523.7 > Genes < Gm48091-201lncRNA (Comprehensive set... Regulatory Build 27.8Mb 28.0Mb 28.2Mb 28.4Mb 28.6Mb 28.8Mb Reverse strand 1.15 Mb 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 RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000115243 1.13 Mb Forward strand Opcml-202 >protein coding ENSMUSP00000110... Transmembrane heli... Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily SMART Immunoglobulin subtype Immunoglobulin subtype 2 Pfam PF13927 Immunoglobulin I-set PROSITE profiles Immunoglobulin-like domain PIRSF PIRSF000615 PANTHER PTHR42757 PTHR42757:SF9 Gene3D Immunoglobulin-like fold CDD cd00096 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 240 280 337 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.