https://www.alphaknockout.com

Mouse Adamts6 Knockout Project (CRISPR/Cas9)

Objective: To create a Adamts6 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Adamts6 gene (NCBI Reference Sequence: NM_001081020 ; Ensembl: ENSMUSG00000046169 ) is located on Mouse chromosome 13. 25 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 25 (Transcript: ENSMUST00000065766). Exon 2~3 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: Mice homozygous for induced mutations exhibit cardiovascular defects including double outlet right ventricle, ventricular septal defects and biventricular hypertrophy, and hydrops, thymus hypoplasia short snout and cleft palate.

Exon 2 starts from the coding region. Exon 2~3 covers 13.79% of the coding region. The size of effective KO region: ~3080 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 25

Legends Exon of mouse Adamts6 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 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(30.45% 609) | C(15.7% 314) | T(38.9% 778) | G(14.95% 299)

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(2000bp) | A(25.1% 502) | C(17.3% 346) | T(38.35% 767) | G(19.25% 385)

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% chr13 + 104292729 104294728 2000

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 2000 1 2000 2000 100.0% chr13 + 104297528 104299527 2000 browser details YourSeq 232 165 468 2000 90.2% chr13 + 119759324 119759622 299 browser details YourSeq 103 1319 1715 2000 92.7% chr18 - 34878271 35091882 213612 browser details YourSeq 93 1318 1689 2000 77.0% chr19 - 46498235 46498565 331 browser details YourSeq 89 1347 1715 2000 75.2% chr19 + 26763625 26763951 327 browser details YourSeq 88 1318 1715 2000 78.5% chr12 - 75063612 75063978 367 browser details YourSeq 86 1313 1724 2000 89.8% chr12 - 117547154 117555698 8545 browser details YourSeq 84 1499 1714 2000 70.3% chr15 - 41991089 41991274 186 browser details YourSeq 77 1318 1714 2000 71.9% chr12 - 117171336 117171710 375 browser details YourSeq 76 1318 1715 2000 80.0% chr2 - 163444442 163444842 401 browser details YourSeq 76 1318 1715 2000 75.8% chr4 + 151589613 151589955 343 browser details YourSeq 76 1324 1714 2000 72.6% chr10 + 127122786 127123126 341 browser details YourSeq 73 1318 1715 2000 91.1% chr10 - 58844296 58844873 578 browser details YourSeq 73 1318 1715 2000 76.3% chr19 + 11109585 11109947 363 browser details YourSeq 73 1318 1728 2000 81.6% chr1 + 88829198 88829625 428 browser details YourSeq 72 1525 1712 2000 79.8% chr12 - 99566319 99566468 150 browser details YourSeq 70 1514 1683 2000 72.9% chr2 + 157723576 157723711 136 browser details YourSeq 68 1581 1726 2000 81.0% chr5 + 36859019 36859156 138 browser details YourSeq 67 1516 1715 2000 81.9% chrX - 163415734 163415916 183 browser details YourSeq 67 1313 1715 2000 81.8% chr6 + 51254664 51255058 395

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: Adamts6 a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 6 [ Mus musculus (house mouse) ] Gene ID: 108154, updated on 12-Aug-2019

Gene summary

Official Symbol Adamts6 provided by MGI Official Full Name a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 6 provided by MGI Primary source MGI:MGI:1347348 See related Ensembl:ENSMUSG00000046169 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 ADAM-TS6; b2b2029Clo; b2b2182Clo; b2b2228Clo; b2b1879.1Clo; b2b2187.1Clo; A930019D11Rik Expression Broad expression in limb E14.5 (RPKM 4.5), bladder adult (RPKM 3.5) and 21 other tissues See more Orthologs human all

Genomic context

Location: 13; 13 D1 See Adamts6 in Genome Data Viewer Exon count: 28

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (104286518..104496695)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (105077953..105284843)

Chromosome 13 - NC_000079.6

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

Transcript information: This gene has 7 transcripts

Gene: Adamts6 ENSMUSG00000046169

Description a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 6 [Source:MGI Symbol;Acc:MGI:1347348] Gene Synonyms A930019D11Rik, ADAM-TS6, b2b1879.1Clo, b2b2029Clo, b2b2182Clo, b2b2187.1Clo, b2b2228Clo Location Chromosome 13: 104,287,835-104,496,695 forward strand. GRCm38:CM001006.2 About this gene This gene has 7 transcripts (splice variants), 214 orthologues, 25 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

Adamts6- ENSMUST00000224208.1 6480 1117aa ENSMUSP00000153359.1 Protein coding CCDS36773 D3Z1A5 GENCODE 203 basic APPRIS P1

Adamts6- ENSMUST00000065766.6 4822 1117aa ENSMUSP00000064570.6 Protein coding CCDS36773 D3Z1A5 TSL:5 201 GENCODE basic APPRIS P1

Adamts6- ENSMUST00000224784.1 7690 468aa ENSMUSP00000153665.1 Nonsense mediated - A0A286YEC9 - 207 decay

Adamts6- ENSMUST00000224742.1 4901 292aa ENSMUSP00000152936.1 Nonsense mediated - A0A286YCD8 - 206 decay

Adamts6- ENSMUST00000224303.1 4672 292aa ENSMUSP00000153431.1 Nonsense mediated - A0A286YCD8 - 204 decay

Adamts6- ENSMUST00000223562.1 3840 468aa ENSMUSP00000153504.1 Nonsense mediated - A0A286YEC9 - 202 decay

Adamts6- ENSMUST00000224504.1 745 96aa ENSMUSP00000153194.1 Nonsense mediated - A0A286YCZ1 CDS 5' 205 decay incomplete

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

228.86 kb Forward strand

104.3Mb 104.4Mb 104.5Mb Genes (Comprehensive set... Adamts6-207 >nonsense mediated decay

Adamts6-202 >nonsense mediated decay

Adamts6-201 >protein coding

Adamts6-206 >nonsense mediated decay

Adamts6-203 >protein coding

Adamts6-204 >nonsense mediated decay

Adamts6-205 >nonsense mediated decay

Contigs < AC165950.1 < AC154830.2 Regulatory Build

104.3Mb 104.4Mb 104.5Mb Reverse strand 228.86 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding

Non-Protein Coding

processed transcript

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

Transcript: ENSMUST00000065766

206.89 kb Forward strand

Adamts6-201 >protein coding

ENSMUSP00000064... Cleavage site (Sign... Superfamily SSF55486 Thrombospondin type-1 (TSP1) repeat superfamily SMART Thrombospondin type-1 (TSP1) repeat Prints ADAMTS/ADAMTS-like Pfam Peptidase M12B, ADAM/reprolysin Thrombospondin type-1 (TSP1) repeat PLAC

Peptidase M12B, propeptide ADAM-TS Spacer 1

ADAM cysteine-rich domain 2 PROSITE profiles Peptidase M12B, ADAM/reprolysin Thrombospondin type-1 (TSP1) repeat PLAC

PANTHER PTHR13723:SF27

PTHR13723 Gene3D Metallopeptidase, catalytic domain superfamily 2.60.120.830

Thrombospondin type-1 (TSP1) repeat superfamily CDD cd04273

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

Variant Legend stop gained missense variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1000 1117

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

Page 9 of 9