https://www.alphaknockout.com

Mouse Gpnmb Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Gpnmb gene (NCBI Reference Sequence: NM_053110 ; Ensembl: ENSMUSG00000029816 ) is located on Mouse chromosome 6. 11 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 11 (Transcript: ENSMUST00000031840). Exon 2~8 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 mutants exhibit dispersed pigmentation of the iris, deterioration of the posterior iris epithelium and slit- like transillumination defects. The mutation contributes to glaucoma, especially in combination with the brown coat color mutation.

Exon 2 starts from about 4.12% of the coding region. Exon 2~8 covers 69.57% of the coding region. The size of effective KO region: ~8360 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 5' gRNA region gRNA region 3'

1 2 3 4 5 6 7 8 11

Legends Exon of mouse Gpnmb 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 2 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 691 bp section downstream of Exon 8 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(30.7% 614) | C(21.75% 435) | T(27.2% 544) | G(20.35% 407)

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(691bp) | A(32.27% 223) | C(20.69% 143) | T(23.59% 163) | G(23.44% 162)

Note: The 691 bp section downstream of Exon 8 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% chr6 + 49040768 49042767 2000 browser details YourSeq 249 398 890 2000 89.1% chr15 - 63497850 63907862 410013 browser details YourSeq 249 399 896 2000 86.2% chr9 + 40632972 40633400 429 browser details YourSeq 248 397 875 2000 90.9% chr12 + 111563463 111563949 487 browser details YourSeq 236 399 869 2000 84.9% chr10 - 120126079 120126370 292 browser details YourSeq 199 397 839 2000 90.0% chr4 - 42994368 42995015 648 browser details YourSeq 199 398 818 2000 84.4% chr17 + 55919515 55919842 328 browser details YourSeq 185 91 577 2000 82.5% chr16 - 56575920 56576285 366 browser details YourSeq 181 91 888 2000 80.8% chr16 + 96115789 96116143 355 browser details YourSeq 175 91 577 2000 82.6% chr15 - 76376661 76377027 367 browser details YourSeq 173 120 576 2000 91.4% chr17 - 7987893 7988496 604 browser details YourSeq 163 91 575 2000 85.0% chr5 - 145277855 145278221 367 browser details YourSeq 163 397 889 2000 84.5% chr7 + 140148497 140148972 476 browser details YourSeq 156 49 582 2000 82.8% chr3 - 9914964 9915159 196 browser details YourSeq 156 125 886 2000 82.9% chr10 + 63369817 63370253 437 browser details YourSeq 152 397 577 2000 89.9% chr7 - 100746265 100746442 178 browser details YourSeq 152 397 576 2000 92.3% chr1 - 71713576 71713755 180 browser details YourSeq 149 366 569 2000 84.3% chr13 + 112902040 112902231 192 browser details YourSeq 148 737 1390 2000 85.2% chr16 + 38677985 38678567 583 browser details YourSeq 147 388 575 2000 87.7% chr17 + 56237255 56237440 186

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 691 1 691 691 100.0% chr6 + 49051128 49051818 691 browser details YourSeq 56 224 611 691 66.7% chr1 - 91544572 91544726 155 browser details YourSeq 45 417 611 691 90.4% chr19 + 5470954 5471147 194 browser details YourSeq 43 275 335 691 86.5% chr4 - 141650177 141650238 62 browser details YourSeq 43 320 590 691 89.1% chr2 + 32738005 32738486 482 browser details YourSeq 42 539 613 691 92.0% chr19 - 5429110 5429186 77 browser details YourSeq 42 321 615 691 60.9% chr1 + 187464958 187465076 119 browser details YourSeq 36 539 610 691 92.9% chr18 - 38995182 38995254 73 browser details YourSeq 36 527 573 691 84.1% chr1 + 170538153 170538197 45 browser details YourSeq 35 250 335 691 88.9% chr18 - 57278675 57278763 89 browser details YourSeq 35 537 585 691 95.0% chr12 - 16779763 16779821 59 browser details YourSeq 35 532 573 691 92.7% chr12 + 13220220 13220261 42 browser details YourSeq 34 274 360 691 90.5% chr5 - 132590116 132590204 89 browser details YourSeq 34 535 575 691 94.8% chr3 - 87548050 87548091 42 browser details YourSeq 34 535 575 691 92.7% chr16 + 30091064 30091105 42 browser details YourSeq 33 538 588 691 82.4% chr6 - 52469303 52469353 51 browser details YourSeq 33 539 576 691 94.6% chr7 + 29031436 29031473 38 browser details YourSeq 32 535 574 691 90.0% chr9 - 76090983 76091022 40 browser details YourSeq 32 539 574 691 94.5% chr3 - 28015258 28015293 36 browser details YourSeq 32 533 574 691 88.1% chr2 - 101930281 101930322 42

Note: The 691 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 https://www.alphaknockout.com

Gene and protein information: Gpnmb glycoprotein (transmembrane) nmb [ Mus musculus (house mouse) ] Gene ID: 93695, updated on 16-Sep-2019

Gene summary

Official Symbol Gpnmb provided by MGI Official Full Name glycoprotein (transmembrane) nmb provided by MGI Primary source MGI:MGI:1934765 See related Ensembl:ENSMUSG00000029816 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 ipd; Dchil; DC-HIL Expression Biased expression in bladder adult (RPKM 24.4), genital fat pad adult (RPKM 18.0) and 8 other tissuesS ee more Orthologs human all

Genomic context

Location: 6 B2.3; 6 23.82 cM See Gpnmb in Genome Data Viewer Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (49036518..49058182)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (48986517..49008181)

Chromosome 6 - NC_000072.6

Page 6 of 9 https://www.alphaknockout.com

Transcript information: This gene has 5 transcripts

Gene: Gpnmb ENSMUSG00000029816

Description glycoprotein (transmembrane) nmb [Source:MGI Symbol;Acc:MGI:1934765] Gene Synonyms DC-HIL, Dchil, Osteoactivin Location Chromosome 6: 49,036,546-49,070,929 forward strand. GRCm38:CM000999.2 About this gene This gene has 5 transcripts (splice variants), 197 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 13 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Gpnmb- ENSMUST00000031840.9 3701 574aa ENSMUSP00000031840.7 Protein coding CCDS20122 Q99P91 TSL:1 201 GENCODE basic APPRIS P2

Gpnmb- ENSMUST00000204260.1 2430 526aa ENSMUSP00000145376.1 Protein coding - Q8BVA0 TSL:1 203 GENCODE basic APPRIS ALT2

Gpnmb- ENSMUST00000203757.1 365 37aa ENSMUSP00000145090.1 Nonsense mediated - A0A0N4SVG5 TSL:2 202 decay

Gpnmb- ENSMUST00000205103.1 1832 No - Retained intron - - TSL:NA 205 protein

Gpnmb- ENSMUST00000204460.1 645 No - Retained intron - - TSL:1 204 protein

Page 7 of 9 https://www.alphaknockout.com

54.38 kb Forward strand

49.03Mb 49.04Mb 49.05Mb 49.06Mb 49.07Mb 49.08Mb Genes (Comprehensive set... Gpnmb-205 >retained intron Gpnmb-204 >retained intron Malsu1-202 >nonsense mediated decay

Gpnmb-201 >protein coding Gm19253-201 >processed pseudogene

Gpnmb-202 >nonsense mediated decay Malsu1-203 >protein coding

Gpnmb-203 >protein coding Malsu1-201 >lncRNA

Malsu1-205 >lncRNA

Contigs AC153620.4 >

Genes < Gm7890-201processed pseudogene (Comprehensive set...

Regulatory Build

49.03Mb 49.04Mb 49.05Mb 49.06Mb 49.07Mb 49.08Mb Reverse strand 54.38 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 RNA gene pseudogene

Page 8 of 9 https://www.alphaknockout.com

Transcript: ENSMUST00000031840

21.57 kb Forward strand

Gpnmb-201 >protein coding

ENSMUSP00000031... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily PKD domain superfamily

SMART PKD/Chitinase domain Pfam PKD domain PROSITE profiles PKD domain

PANTHER PTHR11861:SF11

PTHR11861 Gene3D Immunoglobulin-like fold

CDD cd00146

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend stop gained missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 574

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

Page 9 of 9