https://www.alphaknockout.com Mouse Cadm3 Knockout Project (CRISPR/Cas9) Objective: To create a Cadm3 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cadm3 gene (NCBI Reference Sequence: NM_053199 ; Ensembl: ENSMUSG00000005338 ) is located on Mouse chromosome 1. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000111220). Exon 2~6 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: Mice homozygous for a null allele exhibit delayed myelination. Other mice with ubiquitous conditional deletion of the gene do not display neurological abnormalities. Exon 2 starts from about 6.99% of the coding region. Exon 2~6 covers 58.42% of the coding region. The size of effective KO region: ~4894 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 2 3 4 5 6 9 Legends Exon of mouse Cadm3 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 2 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 445 bp section downstream 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. 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(26.85% 537) | C(21.05% 421) | T(30.0% 600) | G(22.1% 442) Note: The 2000 bp section upstream of Exon 2 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(445bp) | A(26.29% 117) | C(34.38% 153) | T(26.07% 116) | G(13.26% 59) Note: The 445 bp section downstream 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. 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 - 173346563 173348562 2000 browser details YourSeq 56 1277 1389 2000 87.7% chr1 + 74955129 74955244 116 browser details YourSeq 51 1277 1389 2000 83.9% chr17 - 13002129 13002240 112 browser details YourSeq 49 1278 1389 2000 84.6% chr15 + 78864478 78864722 245 browser details YourSeq 49 1267 1381 2000 84.3% chr15 + 43129644 43129755 112 browser details YourSeq 44 1277 1386 2000 76.0% chr2 + 156564590 156564680 91 browser details YourSeq 43 1269 1368 2000 86.6% chr16 + 60206794 60206892 99 browser details YourSeq 41 982 1380 2000 66.0% chr18 - 34736369 34736682 314 browser details YourSeq 40 1339 1389 2000 90.0% chr14 - 45350011 45350063 53 browser details YourSeq 40 1273 1389 2000 93.4% chr1 - 67545629 67545747 119 browser details YourSeq 39 1055 1303 2000 95.6% chr13 - 84402440 84402946 507 browser details YourSeq 39 923 993 2000 85.8% chr4 + 145321482 145321551 70 browser details YourSeq 38 1348 1389 2000 95.3% chr9 - 57452605 57452646 42 browser details YourSeq 36 1346 1389 2000 91.0% chr18 + 42592916 42592959 44 browser details YourSeq 35 1277 1366 2000 92.9% chr14 - 54593748 54593838 91 browser details YourSeq 35 1277 1364 2000 89.5% chr11 - 115764250 115764336 87 browser details YourSeq 35 1351 1391 2000 92.7% chr11 + 106077074 106077114 41 browser details YourSeq 34 1277 1367 2000 92.5% chr11 + 74545490 74545581 92 browser details YourSeq 32 1267 1307 2000 94.3% chr8 - 61995999 61996043 45 browser details YourSeq 32 779 821 2000 88.9% chr1 - 42442095 42442136 42 Note: The 2000 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 445 1 445 445 100.0% chr1 - 173341224 173341668 445 browser details YourSeq 30 238 386 445 65.7% chr18 + 29927467 29927591 125 browser details YourSeq 23 130 153 445 100.0% chr17 - 82131964 82131996 33 browser details YourSeq 23 374 398 445 87.5% chr1 - 168277815 168277838 24 Note: The 445 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Cadm3 cell adhesion molecule 3 [ Mus musculus (house mouse) ] Gene ID: 94332, updated on 11-Sep-2019 Gene summary Official Symbol Cadm3 provided by MGI Official Full Name cell adhesion molecule 3 provided by MGI Primary source MGI:MGI:2137858 See related Ensembl:ENSMUSG00000005338 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 BIgR; Necl1; Tsll1; Igsf4b; Necl-1; SynCAM3 Expression Biased expression in cerebellum adult (RPKM 149.0), frontal lobe adult (RPKM 64.2) and 7 other tissues See more Orthologs human all Genomic context Location: 1; 1 H3 See Cadm3 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (173331892..173368139, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (175264385..175297826, complement) Chromosome 1 - NC_000067.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Cadm3 ENSMUSG00000005338 Description cell adhesion molecule 3 [Source:MGI Symbol;Acc:MGI:2137858] Gene Synonyms BIgR, Igsf4b, Necl-1, Necl1, SynCAM3, Tsll1 Location Chromosome 1: 173,333,258-173,367,695 reverse strand. GRCm38:CM000994.2 About this gene This gene has 5 transcripts (splice variants), 195 orthologues, 15 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cadm3-202 ENSMUST00000111220.7 5155 396aa ENSMUSP00000106851.1 Protein coding CCDS15528 Q99N28 TSL:1 GENCODE basic APPRIS P2 Cadm3-201 ENSMUST00000005470.4 2518 430aa ENSMUSP00000005470.4 Protein coding - K4DI58 TSL:5 GENCODE basic APPRIS ALT1 Cadm3-205 ENSMUST00000157032.7 2746 No protein - Retained intron - - TSL:1 Cadm3-204 ENSMUST00000136540.1 855 No protein - Retained intron - - TSL:1 Cadm3-203 ENSMUST00000126963.1 774 No protein - Retained intron - - TSL:3 54.44 kb Forward strand 173.33Mb 173.34Mb 173.35Mb 173.36Mb 173.37Mb Genes Aim2-202 >protein coding (Comprehensive set... Contigs < AC087781.17 AC084073.22 > Genes (Comprehensive set... < Ackr1-203lncRNA < Cadm3-205retained intron < Ackr1-201protein coding < Cadm3-203retained intron < Ackr1-202protein coding < Cadm3-202protein coding < Cadm3-201protein coding < Cadm3-204retained intron Regulatory Build 173.33Mb 173.34Mb 173.35Mb 173.36Mb 173.37Mb Reverse strand 54.44 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000111220 < Cadm3-202protein coding Reverse strand 34.44 kb ENSMUSP00000106... Transmembrane heli... PDB-ENSP mappings Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily SMART Immunoglobulin subtype 2 Neurexin/syndecan/glycophorin C Immunoglobulin subtype Pfam Immunoglobulin I-set CD80-like, immunoglobulin C2-set PF13927 PROSITE profiles Immunoglobulin-like domain PANTHER PTHR45889 PTHR45889:SF5 Gene3D Immunoglobulin-like fold CDD cd05882 cd07705 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 396 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.