https://www.alphaknockout.com

Mouse Xpo4 Knockout Project (CRISPR/Cas9)

Objective: To create a Xpo4 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Xpo4 gene (NCBI Reference Sequence: NM_020506 ; Ensembl: ENSMUSG00000021952 ) is located on Mouse chromosome 14. 23 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 23 (Transcript: ENSMUST00000174545). Exon 4~6 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 a gene trapped allele appear phenotypically normal.

Exon 4 starts from about 9.21% of the coding region. Exon 4~6 covers 11.87% of the coding region. The size of effective KO region: ~9001 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 5 6 23

Legends Exon of mouse Xpo4 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 4 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 6 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(27.6% 552) | C(16.45% 329) | T(33.25% 665) | G(22.7% 454)

Note: The 2000 bp section upstream of Exon 4 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(23.65% 473) | C(18.95% 379) | T(35.65% 713) | G(21.75% 435)

Note: The 2000 bp section downstream of Exon 6 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr14 - 57638358 57640357 2000 browser details YourSeq 63 6 287 2000 70.4% chr10 + 95827659 95827789 131 browser details YourSeq 58 6 96 2000 84.6% chr11 - 62043841 62043928 88 browser details YourSeq 57 12 90 2000 88.0% chr1 - 153179470 153179550 81 browser details YourSeq 56 6 103 2000 78.6% chr11 - 115548501 115548598 98 browser details YourSeq 55 10 86 2000 85.8% chr10 - 23802574 23802650 77 browser details YourSeq 55 6 90 2000 82.4% chr13 + 93333545 93333629 85 browser details YourSeq 54 11 90 2000 93.5% chr6 - 82721152 82721233 82 browser details YourSeq 54 1 81 2000 85.0% chr3 - 90599039 90599183 145 browser details YourSeq 52 1 90 2000 78.9% chr14 - 22791311 22791400 90 browser details YourSeq 51 6 90 2000 80.0% chr11 + 105208283 105208367 85 browser details YourSeq 50 6 77 2000 84.8% chr9 + 117802621 117802692 72 browser details YourSeq 50 5 90 2000 84.8% chr19 + 45491967 45492703 737 browser details YourSeq 50 13 104 2000 77.2% chr11 + 58600936 58601027 92 browser details YourSeq 49 2 76 2000 85.6% chr16 - 9902553 9902629 77 browser details YourSeq 49 11 90 2000 83.8% chr14 + 115454199 115454476 278 browser details YourSeq 48 1 90 2000 80.6% chr12 + 77832847 77832937 91 browser details YourSeq 48 6 84 2000 80.8% chr12 + 65246978 65247252 275 browser details YourSeq 47 26 90 2000 86.2% chr9 - 75630282 75630346 65 browser details YourSeq 47 11 89 2000 79.8% chr2 - 173337352 173337430 79

Note: The 2000 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 2000 1 2000 2000 100.0% chr14 - 57627357 57629356 2000 browser details YourSeq 230 1276 1895 2000 90.6% chr3 - 94740486 95056162 315677 browser details YourSeq 209 1301 1900 2000 88.9% chr7 + 35433371 35912344 478974 browser details YourSeq 195 1310 1890 2000 82.4% chr5 - 123904680 123905003 324 browser details YourSeq 181 1288 1863 2000 81.2% chr7 + 6291359 6291718 360 browser details YourSeq 177 1164 1635 2000 88.9% chr2 + 180252938 180253361 424 browser details YourSeq 175 1164 1463 2000 95.9% chr4 + 32925195 32925835 641 browser details YourSeq 171 1288 1891 2000 83.3% chr19 - 24550175 24550697 523 browser details YourSeq 163 1297 1895 2000 79.6% chr2 - 155048238 155048619 382 browser details YourSeq 162 1279 1449 2000 97.7% chr2 - 29545866 29546038 173 browser details YourSeq 160 1279 1463 2000 92.6% chr17 - 47525661 47525841 181 browser details YourSeq 160 1288 1460 2000 97.7% chr13 - 110958733 110958926 194 browser details YourSeq 157 1288 1559 2000 94.3% chr2 + 121973799 121974069 271 browser details YourSeq 157 1289 1463 2000 94.9% chr19 + 20398104 20398278 175 browser details YourSeq 157 1293 1639 2000 87.2% chr11 + 62522799 62522971 173 browser details YourSeq 157 1288 1452 2000 97.6% chr11 + 31031688 31031852 165 browser details YourSeq 156 1288 1462 2000 94.7% chr13 - 54535752 54535924 173 browser details YourSeq 156 1282 1654 2000 86.1% chr6 + 113530378 113530560 183 browser details YourSeq 155 1277 1454 2000 91.5% chr14 - 18368728 18368902 175 browser details YourSeq 155 1295 1635 2000 87.5% chr10 - 126894751 126894927 177

Note: The 2000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Xpo4 exportin 4 [ Mus musculus (house mouse) ] Gene ID: 57258, updated on 15-Aug-2019

Gene summary

Official Symbol Xpo4 provided by MGI Official Full Name exportin 4 provided by MGI Primary source MGI:MGI:1888526 See related Ensembl:ENSMUSG00000021952 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 mKIAA1721; B430309A01Rik Expression Ubiquitous expression in limb E14.5 (RPKM 4.9), CNS E11.5 (RPKM 4.9) and 27 other tissues See more Orthologs human all

Genomic context

Location: 14; 14 C3 See Xpo4 in Genome Data Viewer Exon count: 24

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (57577521..57668232, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (58201108..58283793, complement)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 9 transcripts

Gene: Xpo4 ENSMUSG00000021952

Description exportin 4 [Source:MGI Symbol;Acc:MGI:1888526] Gene Synonyms B430309A01Rik Location Chromosome 14: 57,577,521-57,665,430 reverse strand. GRCm38:CM001007.2 About this gene This gene has 9 transcripts (splice variants), 211 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Xpo4- ENSMUST00000174545.8 8680 1151aa ENSMUSP00000133280.1 Protein coding CCDS56961 A0A0R4J254 TSL:1 209 GENCODE basic APPRIS P2

Xpo4- ENSMUST00000089482.11 3456 1151aa ENSMUSP00000086909.5 Protein coding - Q9ESJ0 TSL:5 201 GENCODE basic APPRIS ALT1

Xpo4- ENSMUST00000174152.7 2323 70aa ENSMUSP00000133497.1 Nonsense mediated - G3UX04 TSL:1 208 decay

Xpo4- ENSMUST00000172524.1 1078 70aa ENSMUSP00000134219.1 Nonsense mediated - G3UX04 TSL:1 202 decay

Xpo4- ENSMUST00000173940.7 1893 No - Retained intron - - TSL:1 207 protein

Xpo4- ENSMUST00000173172.1 1765 No - Retained intron - - TSL:1 205 protein

Xpo4- ENSMUST00000172647.1 947 No - Retained intron - - TSL:3 204 protein

Xpo4- ENSMUST00000173638.1 665 No - Retained intron - - TSL:2 206 protein

Xpo4- ENSMUST00000172539.7 707 No - lncRNA - - TSL:2 203 protein

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107.91 kb Forward strand 57.58Mb 57.60Mb 57.62Mb 57.64Mb 57.66Mb Genes 1700039M10Rik-201 >lncRNA (Comprehensive set...

Contigs AC154675.2 > < AC154504.2 Genes (Comprehensive set... < Eef1akmt1-203protein coding < Xpo4-208nonsense mediated decay

< Eef1akmt1-201protein coding < Xpo4-203lncRNA

< Eef1akmt1-202lncRNA < Xpo4-207retained intron

< Xpo4-209protein coding

< Xpo4-201protein coding

< Xpo4-205retained intron

< Xpo4-202nonsense mediated decay

< Gm49361-201nonsense mediated decay

< Xpo4-204retained intron

< Xpo4-206retained intron

Regulatory Build

57.58Mb 57.60Mb 57.62Mb 57.64Mb 57.66Mb Reverse strand 107.91 kb

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

Gene Legend Protein Coding

Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000174545

< Xpo4-209protein coding

Reverse strand 87.91 kb

ENSMUSP00000133... Low complexity (Seg) Superfamily Armadillo-type fold Pfam Exportin-1, C-terminal PANTHER PTHR12596

PTHR12596:SF1 Gene3D Armadillo-like helical

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1000 1151

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

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