https://www.alphaknockout.com

Mouse Maged1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Maged1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Maged1 (NCBI Reference Sequence: NM_019791 ; Ensembl: ENSMUSG00000025151 ) is located on Mouse X. 13 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 12 (Transcript: ENSMUST00000026142). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Maged1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-206L10 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: 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 100% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 567 bp, and the size of intron 3 for 3'-loxP site insertion: 573 bp. The size of effective cKO region: ~1941 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 5' gRNA region gRNA region 3'

1 2 3 4 5 6 7 8 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Maged1 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. 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 GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8441bp) | A(24.78% 2092) | C(26.51% 2238) | T(24.93% 2104) | G(23.78% 2007)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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 - 94541546 94544545 3000 browser details YourSeq 530 674 1209 3000 99.5% chrX - 94507913 94508448 536 browser details YourSeq 56 577 678 3000 87.5% chr15 - 71199032 71199130 99 browser details YourSeq 49 587 643 3000 96.3% chr3 + 48893693 48893753 61 browser details YourSeq 48 591 655 3000 94.6% chr2 + 79322299 79322370 72 browser details YourSeq 46 585 647 3000 94.4% chr9 - 112946651 112946718 68 browser details YourSeq 46 585 638 3000 84.0% chr5 - 58001195 58001244 50 browser details YourSeq 46 579 638 3000 86.6% chr10 - 64481438 64481495 58 browser details YourSeq 46 590 643 3000 92.6% chrX + 94543903 94543956 54 browser details YourSeq 46 585 638 3000 84.0% chr5 + 58001187 58001236 50 browser details YourSeq 45 584 642 3000 96.0% chr2 - 79322299 79322358 60 browser details YourSeq 45 585 638 3000 94.0% chr2 - 66826249 66826304 56 browser details YourSeq 45 588 643 3000 92.5% chr12 - 54462539 54462620 82 browser details YourSeq 44 1626 1768 3000 70.9% chr8 - 33932311 33932392 82 browser details YourSeq 44 585 643 3000 85.5% chr3 - 48893728 48893782 55 browser details YourSeq 43 585 641 3000 87.5% chr8 - 12526325 12526379 55 browser details YourSeq 43 585 638 3000 95.8% chr15 - 25255532 25255585 54 browser details YourSeq 42 585 640 3000 78.8% chr12 - 107092959 107093006 48 browser details YourSeq 42 575 639 3000 91.9% chr3 + 57298487 57298551 65 browser details YourSeq 42 575 635 3000 92.0% chr11 + 47265986 47266052 67

Note: The 3000 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 3000 1 3000 3000 100.0% chrX - 94536605 94539604 3000 browser details YourSeq 236 969 2717 3000 85.1% chrX - 150650448 150653321 2874 browser details YourSeq 37 2857 2913 3000 82.5% chr13 + 107046186 107046242 57 browser details YourSeq 36 2859 2900 3000 92.9% chr7 + 141052360 141052401 42 browser details YourSeq 34 2870 2908 3000 86.5% chr19 + 10327470 10327506 37 browser details YourSeq 31 2859 2907 3000 91.0% chr10 + 9585372 9585419 48 browser details YourSeq 28 2880 2913 3000 91.2% chr6 - 38090308 38090341 34 browser details YourSeq 28 2860 2916 3000 96.7% chr2 + 4830725 4830782 58 browser details YourSeq 27 2537 2565 3000 96.6% chr19 - 54730855 54730883 29 browser details YourSeq 27 134 160 3000 100.0% chr5 + 83402631 83402657 27 browser details YourSeq 26 134 159 3000 100.0% chr6 + 118966341 118966366 26 browser details YourSeq 25 2880 2906 3000 96.3% chr18 - 66350780 66350806 27 browser details YourSeq 25 2892 2944 3000 73.6% chr18 + 81935805 81935857 53

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 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 5 of 7 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 > (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 6 of 7 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.

Page 7 of 7