http://www.alphaknockout.com/ Mouse Fxyd5 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Fxyd5 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fxyd5 gene ( NCBI Reference Sequence: NM_001287217 ; Ensembl: ENSMUSG00000009687 ) is located on mouse chromosome 7. 9 exons are identified , with the ATG start codon in exon 2 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000009831). Exon 4~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the mouse Fxyd5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-327F20 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:

Exon 4~8 is not frameshift exon, and covers 65.92% of the coding region. The size of intron 3 for 5'-loxP site insertion: 1088 bp, and the size of intron 8 for 3'-loxP site insertion: 2190 bp. The size of effective cKO region: ~4385 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level. The transcript Fxyd5-212 may be affected by inserting the second loxP.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 3 4 5 6 7 8 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Fxyd5 Homology arm cKO region loxP site

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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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(10886bp) | A(23.45% 2553) | C(25.52% 2778) | G(26.34% 2867) | T(24.69% 2688)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr7 - 31039345 31042344 3000 browser details YourSeq 145 2180 2362 3000 91.0% chr1 + 185274464 185274663 200 browser details YourSeq 140 2178 2374 3000 87.5% chr16 + 38566757 38566950 194 browser details YourSeq 135 2180 2356 3000 93.1% chr3 + 51575320 51575506 187 browser details YourSeq 134 2178 2362 3000 92.4% chr14 + 62900920 62901104 185 browser details YourSeq 132 2180 2360 3000 91.3% chr11 + 100391638 100391831 194 browser details YourSeq 131 2183 2340 3000 92.9% chrX + 36775949 36776113 165 browser details YourSeq 130 2180 2360 3000 87.4% chr11 - 58108976 58109165 190 browser details YourSeq 130 2179 2351 3000 92.3% chr9 + 88349518 88349696 179 browser details YourSeq 129 2179 2370 3000 88.6% chr13 - 9294612 9294811 200 browser details YourSeq 129 2180 2360 3000 90.2% chr1 + 170984082 170984269 188 browser details YourSeq 127 2179 2362 3000 90.0% chr2 + 53911740 53911931 192 browser details YourSeq 125 2169 2356 3000 89.4% chr12 - 13954638 13954829 192 browser details YourSeq 124 2180 2356 3000 92.0% chr7 + 141072983 141073312 330 browser details YourSeq 121 2182 2355 3000 90.7% chr18 - 42071383 42071566 184 browser details YourSeq 121 2180 2355 3000 90.2% chr17 + 33514805 33514990 186 browser details YourSeq 120 2180 2316 3000 94.2% chr5 - 66382701 66383036 336 browser details YourSeq 120 2180 2323 3000 92.9% chr4 + 100602728 100602883 156 browser details YourSeq 119 2180 2367 3000 88.9% chr14 + 31164658 31164849 192 browser details YourSeq 119 2184 2346 3000 90.5% chr13 + 43318738 43318907 170

Note: The 3000 bp section upstream of Exon 4 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% chr7 - 31031959 31034958 3000 browser details YourSeq 105 1541 1708 3000 87.2% chr10 + 66997478 67176005 178528 browser details YourSeq 76 1574 1710 3000 77.8% chr11 - 120797632 120797813 182 browser details YourSeq 74 164 543 3000 71.1% chr18 + 42104604 42104855 252 browser details YourSeq 72 1552 1651 3000 82.2% chr9 + 106284323 106284417 95 browser details YourSeq 70 1460 1809 3000 78.7% chr4 - 136798958 136799186 229 browser details YourSeq 70 326 540 3000 79.0% chr14 - 24513511 24513647 137 browser details YourSeq 70 1455 1603 3000 96.1% chr4 + 123442363 123442852 490 browser details YourSeq 66 158 366 3000 92.5% chr15 - 98612894 98613188 295 browser details YourSeq 65 1584 1681 3000 83.7% chr7 - 35308621 35308719 99 browser details YourSeq 61 1564 1649 3000 85.9% chr1 - 93684298 93684387 90 browser details YourSeq 61 1539 1708 3000 89.7% chrX + 149051040 149051514 475 browser details YourSeq 61 166 543 3000 69.2% chr4 + 142244775 142244869 95 browser details YourSeq 61 339 536 3000 95.6% chr4 + 136853287 136853747 461 browser details YourSeq 60 1492 1609 3000 92.8% chr3 + 88841239 88841417 179 browser details YourSeq 58 196 362 3000 90.3% chr8 - 118519478 118519745 268 browser details YourSeq 57 324 540 3000 91.4% chr16 + 16228888 16229361 474 browser details YourSeq 57 1504 1807 3000 65.1% chr11 + 82516093 82516209 117 browser details YourSeq 56 1542 1653 3000 71.2% chr10 - 86756331 86756432 102 browser details YourSeq 55 307 544 3000 95.1% chr11 + 78985748 78986076 329

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 http://www.alphaknockout.com/ Gene and protein information: Fxyd5 FXYD domain-containing ion transport regulator 5 [ Mus musculus (house mouse) ] Gene ID: 18301, updated on 25-Sep-2020

Gene summary

Official Symbol Fxyd5 provided by MGI Official Full Name FXYD domain-containing ion transport regulator 5 provided by MGI Primary source MGI:MGI:1201785 See related Ensembl:ENSMUSG00000009687 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Oi; RIC; EF-8; Oit2 Summary This gene encodes a precursor protein that is member of the FXYD family of transmembrane glycoproteins. Like most Expression members of the FXYD family, the encoded protein is a subunit of the sodium-potassium adenosine triphosphatase pump. FXYD family members have tissue-specific expression and differentially regulate the activity of this pump. The protein encoded by this gene also plays a role in cell adhesion and motility. The orthologous human protein inhibits epithelial cadherin, a calcium-dependent adhesion protein and is associated with cancer (promotes metastasis). Alternative splicing of this mouse gene results in multiple transcript variants. [provided by RefSeq, Dec 2013] Orthologs Broad expression in thymus adult (RPKM 35.8), spleen adult (RPKM 33.6) and 24 other tissuesS ee more human all

Genomic context

Location: 7; 7 B1 See Fxyd5 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

109 current GRCm39 (GCF_000001635.27) 7 NC_000073.7 (30732148..30741832, complement)

108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (31032723..31042331, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (31817742..31827341, complement)

Chromosome 7 - NC_000073.7

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Transcript information: This gene has 14 transcripts

Gene: Fxyd5 ENSMUSG00000009687

Description FXYD domain-containing ion transport regulator 5 [Source:MGI Symbol;Acc:MGI:1201785] Gene Synonyms EF-8, Oit2 Location Chromosome 7: 31,032,722-31,042,481 reverse strand. GRCm38:CM001000.2 About this gene This gene has 14 transcripts (splice variants) and 12 orthologues. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Match Flags

Fxyd5- ENSMUST00000161684.7 1172 178aa ENSMUSP00000125285.1 Protein coding CCDS21121 P97808 TSL:1 207 Q3TDW1 GENCODE basic APPRIS P3

Fxyd5- ENSMUST00000161805.7 1166 178aa ENSMUSP00000125398.1 Protein coding CCDS21121 P97808 TSL:1 208 Q3TDW1 GENCODE basic APPRIS P3

Fxyd5- ENSMUST00000162116.7 817 178aa ENSMUSP00000124203.1 Protein coding CCDS21121 P97808 TSL:5 210 Q3TDW1 GENCODE basic APPRIS P3

Fxyd5- ENSMUST00000159924.7 805 178aa ENSMUSP00000124219.1 Protein coding CCDS21121 P97808 TSL:5 203 Q3TDW1 GENCODE basic APPRIS P3

Fxyd5- ENSMUST00000009831.13 803 179aa ENSMUSP00000009831.7 Protein coding CCDS71935 F8WJA1 TSL:3 201 GENCODE basic APPRIS ALT2

Fxyd5- ENSMUST00000162087.7 762 178aa ENSMUSP00000125065.1 Protein coding CCDS21121 P97808 TSL:3 209 Q3TDW1 GENCODE basic APPRIS P3

Fxyd5- ENSMUST00000162733.7 2998 185aa ENSMUSP00000125173.1 Protein coding - E0CXN1 TSL:1 212 GENCODE basic

Fxyd5- ENSMUST00000162250.7 500 127aa ENSMUSP00000124129.1 Protein coding - F6TWM7 CDS 5' 211 incomplete TSL:5

Fxyd5- ENSMUST00000159753.1 375 111aa ENSMUSP00000123813.1 Protein coding - E0CYX7 CDS 3' 202 incomplete TSL:5

Fxyd5- ENSMUST00000160689.7 375 117aa ENSMUSP00000125187.1 Protein coding - E0CXM5 CDS 3' 205 incomplete TSL:3

Fxyd5- ENSMUST00000202395.3 353 108aa ENSMUSP00000144377.1 Protein coding - A0A0J9YUX0 CDS 3' 213 incomplete TSL:3

Fxyd5- ENSMUST00000160006.1 885 No - Retained - - TSL:3 204 protein intron

Fxyd5- ENSMUST00000205301.1 593 No - Retained - - TSL:2 214 protein intron

Fxyd5- ENSMUST00000161259.1 338 No - Retained - - TSL:2 206 protein intron

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29.76 kb Forward strand 31.03Mb 31.04Mb 31.05Mb Contigs < AC149055.6 Genes (Comprehensive set... < Fxyd5-208protein coding < Fxyd7-201protein coding

< Fxyd5-207protein coding < Fxyd7-202protein coding

< Fxyd5-212protein coding < Fxyd1-209retained intron

< Fxyd5-204retained intron < Fxyd5-206retained intron < Fxyd1-213retained intron

< Fxyd5-211protein coding < Mir7050-201miRNA < Fxyd1-206retained intron

< Fxyd5-214retained intron < Fxyd1-208protein coding

< Fxyd5-203protein coding < Fxyd1-214protein coding

< Fxyd5-210protein coding < Fxyd1-212protein coding

< Fxyd5-201protein coding < Fxyd1-210protein coding

< Fxyd5-209protein coding < Fxyd1-201protein coding

< Fxyd5-213protein coding < Fxyd1-211protein coding

< Fxyd5-205protein coding < Fxyd1-203protein coding

< Fxyd5-202protein coding < Fxyd1-207protein coding

< Fxyd1-217protein coding

< Fxyd1-215protein coding

< Fxyd1-202protein coding

< Fxyd1-205protein coding

Regulatory Build

31.03Mb 31.04Mb 31.05Mb Reverse strand 29.76 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000009831

< Fxyd5-201protein coding

Reverse strand 9.38 kb

ENSMUSP00000009... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Pfam Ion-transport regulator, FXYD motif PROSITE patterns Ion-transport regulator, FXYD motif PANTHER PTHR14132

PTHR14132:SF14 Gene3D 1.20.5.780

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 179

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

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