https://www.alphaknockout.com Mouse Maged1 Knockout Project (CRISPR/Cas9) Objective: To create a Maged1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Maged1 gene (NCBI Reference Sequence: NM_019791 ; Ensembl: ENSMUSG00000025151 ) is located on Mouse chromosome X. 13 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 12 (Transcript: ENSMUST00000026142). Exon 2~12 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: Homozygous null mice display defects in apoptotic signaling affecting hair cycling and neuronal physiology. Mice homozygous for a different knock-out allele exhibit hypoactivity, decreased exploration, social withdrawal, anhedonia, behavioral despair andaltered serotonin levels. Exon 2 starts from about 0.04% of the coding region. Exon 2~12 covers 100.0% of the coding region. The size of effective KO region: ~5423 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 7 8 9 10 11 12 13 Legends Exon of mouse Maged1 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 start codon 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 stop codon 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(20.8% 416) | C(29.85% 597) | T(21.95% 439) | G(27.4% 548) Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(31.05% 621) | C(17.45% 349) | T(23.0% 460) | G(28.5% 570) Note: The 2000 bp section downstream of stop codon 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% chrX - 94541296 94543295 2000 browser details YourSeq 44 376 518 2000 70.9% chr8 - 33932311 33932392 82 browser details YourSeq 40 376 463 2000 95.5% chr1 - 118365918 118366047 130 browser details YourSeq 40 226 392 2000 91.7% chr1 - 14715966 14716181 216 browser details YourSeq 35 223 390 2000 76.4% chr1 + 114632490 114632642 153 browser details YourSeq 32 375 425 2000 94.3% chr1 - 64505629 64505683 55 browser details YourSeq 32 376 424 2000 97.1% chr1 - 36147698 36147757 60 browser details YourSeq 30 377 425 2000 94.0% chr1 + 74259291 74259343 53 browser details YourSeq 29 376 425 2000 96.8% chr1 - 86454115 86454167 53 browser details YourSeq 29 376 425 2000 96.8% chr1 + 64548560 64548613 54 browser details YourSeq 29 376 425 2000 96.8% chr1 + 21370980 21371033 54 browser details YourSeq 28 376 425 2000 96.7% chr1 - 60671739 60671792 54 browser details YourSeq 28 376 425 2000 66.7% chr1 - 48565847 48565877 31 browser details YourSeq 25 1024 1058 2000 69.3% chr1 + 98038993 98039018 26 browser details YourSeq 23 1945 1967 2000 100.0% chr7 - 138136735 138136757 23 browser details YourSeq 22 1458 1479 2000 100.0% chr9 - 25766280 25766301 22 browser details YourSeq 22 1724 1747 2000 87.0% chr9 + 84474196 84474218 23 browser details YourSeq 22 335 359 2000 95.9% chr1 + 18744830 18744859 30 browser details YourSeq 21 374 394 2000 100.0% chr1 - 31284109 31284129 21 browser details YourSeq 21 376 396 2000 100.0% chr8 + 84245176 84245196 21 Note: The 2000 bp section upstream of start codon 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% chrX - 94533871 94535870 2000 browser details YourSeq 516 648 1495 2000 88.7% chr11 - 105321522 105322484 963 browser details YourSeq 497 692 1397 2000 90.0% chr13 - 14224252 14225001 750 browser details YourSeq 493 708 1408 2000 90.6% chr1 - 144087807 144088557 751 browser details YourSeq 476 721 1464 2000 89.7% chr17 + 66264782 66265538 757 browser details YourSeq 464 720 1392 2000 89.6% chr11 + 94887508 94888226 719 browser details YourSeq 461 648 1408 2000 88.0% chr2 - 56273844 56274723 880 browser details YourSeq 457 714 1363 2000 90.4% chr5 - 91921474 91922160 687 browser details YourSeq 448 718 1375 2000 89.3% chr1 - 11283568 11284277 710 browser details YourSeq 447 712 1371 2000 87.8% chr2 + 66330271 66330967 697 browser details YourSeq 439 788 1485 2000 89.8% chr10 - 90113278 90363999 250722 browser details YourSeq 439 662 1353 2000 88.2% chrX + 86477506 86478291 786 browser details YourSeq 439 718 1397 2000 88.8% chr1 + 131788506 131789193 688 browser details YourSeq 437 708 1338 2000 88.4% chrX + 150789476 150790144 669 browser details YourSeq 437 742 1494 2000 85.4% chr11 + 33449639 33450378 740 browser details YourSeq 429 712 1408 2000 86.8% chr5 + 18766322 18767065 744 browser details YourSeq 428 713 1312 2000 89.7% chr11 + 3944564 3945204 641 browser details YourSeq 425 712 1338 2000 89.1% chr8 - 57128015 57128688 674 browser details YourSeq 420 735 1334 2000 90.6% chrX - 105137025 105137668 644 browser details YourSeq 417 692 1499 2000 88.6% chr19 + 43344236 43345103 868 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Maged1 melanoma antigen, family D, 1 [ Mus musculus (house mouse) ] Gene ID: 94275, updated on 16-Sep-2019 Gene summary Official Symbol Maged1 provided by MGI Official Full Name melanoma antigen, family D, 1 provided by MGI Primary source MGI:MGI:1930187 See related Ensembl:ENSMUSG00000025151 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 NRAGE; Dlixin; Dlxin1; Dlxin-1; MAGE-D1; DXBwg1492e; 2810433C11Rik; 5430405L04Rik Expression Ubiquitous expression in limb E14.5 (RPKM 316.2), CNS E14 (RPKM 313.7) and 27 other tissues See more Orthologs human all Genomic context Location: X C3; X 41.56 cM See Maged1 in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (94535474..94542025, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (91780813..91787413, complement) Chromosome X - NC_000086.7 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Maged1 ENSMUSG00000025151 Description melanoma antigen, family D, 1 [Source:MGI Symbol;Acc:MGI:1930187] Gene Synonyms 2810433C11Rik, 5430405L04Rik, DXBwg1492e, Dlxin-1, MAGE-D1, Nrage Location Chromosome X: 94,535,474-94,542,143 reverse strand. GRCm38:CM001013.2 About this gene This gene has 7 transcripts (splice variants), 164 orthologues, 32 paralogues, is a member of 1 Ensembl protein family and is associated with 17 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Maged1-201 ENSMUST00000026142.7 2821 775aa ENSMUSP00000026142.7 Protein coding CCDS30280 Q9QYH6 TSL:1 GENCODE basic APPRIS P1 Maged1-207 ENSMUST00000153386.7 2884 No protein - Retained intron - - TSL:1 Maged1-203 ENSMUST00000138146.1 1278 No protein - Retained intron - - TSL:5 Maged1-205 ENSMUST00000139375.7 1274 No protein - Retained intron - - TSL:1 Maged1-202 ENSMUST00000133982.1 943 No protein - Retained intron - - TSL:2 Maged1-206 ENSMUST00000141702.1 873 No protein - Retained intron - - TSL:2 Maged1-204 ENSMUST00000138731.1 779 No protein - Retained intron - - TSL:3 26.67 kb Forward strand 94.53Mb 94.54Mb 94.55Mb Contigs AL627302.20 > AL645466.12 > Genes (Comprehensive set... < Maged1-201protein coding < Gm9005-201processed pseudogene < Maged1-203retained intron < Maged1-202retained intron < Maged1-205retained intron < Maged1-207retained intron < Maged1-204retained intron < Maged1-206retained intron Regulatory Build 94.53Mb 94.54Mb 94.55Mb Reverse strand 26.67 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding pseudogene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000026142 < Maged1-201protein coding Reverse strand 6.67 kb ENSMUSP00000026... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) SMART MAGE homology domain Pfam MAGE homology domain PROSITE profiles MAGE homology domain PANTHER Melanoma-associated antigen Melanoma-associated antigen D1 Gene3D MAGE homology domain, winged helix WH2 motif MAGE homology domain, winged helix WH1 motif All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe insertion missense variant splice region variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 775 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.