https://www.alphaknockout.com

Mouse Ipo5 Knockout Project (CRISPR/Cas9)

Objective: To create a Ipo5 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ipo5 (NCBI Reference Sequence: NM_023579 ; Ensembl: ENSMUSG00000030662 ) is located on Mouse 14. 26 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 26 (Transcript: ENSMUST00000032898). 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:

Exon 2 starts from about 2.77% of the coding region. Exon 2~8 covers 25.01% of the coding region. The size of effective KO region: ~9178 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 2 3 4 5 6 7 8 26

Legends Exon of mouse Ipo5 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 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 1924 bp section downstream of Exon 8 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(26.0% 520) | C(21.2% 424) | T(32.25% 645) | G(20.55% 411)

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(1924bp) | A(31.55% 607) | C(17.78% 342) | T(26.61% 512) | G(24.06% 463)

Note: The 1924 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.

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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 + 120915357 120917356 2000 browser details YourSeq 203 15 1519 2000 93.0% chr11 - 77528372 77819527 291156 browser details YourSeq 164 103 1740 2000 94.6% chr1 - 38066295 38117321 51027 browser details YourSeq 150 1426 1757 2000 90.5% chr8 - 70474701 70670881 196181 browser details YourSeq 116 15 149 2000 95.4% chr7 + 13103459 13103593 135 browser details YourSeq 115 15 149 2000 95.4% chr1 - 13041726 13041869 144 browser details YourSeq 113 16 148 2000 95.3% chr2 - 84970266 84970399 134 browser details YourSeq 113 1463 1757 2000 91.2% chr5 + 110632701 110633236 536 browser details YourSeq 113 15 148 2000 93.3% chr16 + 28248245 28248385 141 browser details YourSeq 113 15 146 2000 95.4% chr1 + 105672830 105672967 138 browser details YourSeq 111 1430 1736 2000 92.4% chr15 + 98133384 98133720 337 browser details YourSeq 110 18 149 2000 94.5% chr9 + 65667205 65667337 133 browser details YourSeq 109 19 145 2000 96.7% chr11 - 95605461 95605592 132 browser details YourSeq 109 1426 1587 2000 85.3% chr10 - 58962776 58962928 153 browser details YourSeq 109 15 149 2000 92.8% chr18 + 56642654 56642787 134 browser details YourSeq 109 18 149 2000 93.8% chr1 + 94335070 94335203 134 browser details YourSeq 108 18 149 2000 93.7% chr10 - 62334171 62334303 133 browser details YourSeq 108 17 148 2000 93.7% chr10 - 41492516 41492656 141 browser details YourSeq 108 1424 1591 2000 83.8% chr8 + 120775036 120775178 143 browser details YourSeq 108 15 158 2000 92.3% chr1 + 81806365 81806510 146

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 1924 1 1924 1924 100.0% chr14 + 120926535 120928458 1924 browser details YourSeq 39 1075 1113 1924 100.0% chr1 - 149354581 149354619 39 browser details YourSeq 32 1299 1337 1924 97.1% chr13 - 38123257 38123297 41 browser details YourSeq 31 1299 1338 1924 87.9% chr11 - 114879294 114879331 38 browser details YourSeq 31 1293 1329 1924 91.9% chr10 + 22471860 22471896 37 browser details YourSeq 30 1299 1329 1924 100.0% chr11 - 76475520 76475553 34 browser details YourSeq 29 1299 1327 1924 100.0% chr10 + 7045449 7045477 29 browser details YourSeq 28 1298 1325 1924 100.0% chr4 - 134972569 134972596 28 browser details YourSeq 28 1298 1325 1924 100.0% chr13 - 101452442 101452469 28 browser details YourSeq 28 1299 1335 1924 76.7% chr13 - 6544391 6544421 31 browser details YourSeq 28 1298 1325 1924 100.0% chr1 - 191018486 191018513 28 browser details YourSeq 27 1299 1327 1924 96.6% chrX - 101403886 101403914 29 browser details YourSeq 27 1299 1325 1924 100.0% chr12 - 20347805 20347831 27 browser details YourSeq 27 1299 1325 1924 100.0% chr12 - 18427729 18427755 27 browser details YourSeq 27 1299 1327 1924 96.6% chr11 - 23204540 23204568 29 browser details YourSeq 27 1301 1327 1924 100.0% chr16 + 22283510 22283536 27 browser details YourSeq 27 1299 1327 1924 96.6% chr1 + 84880983 84881011 29 browser details YourSeq 26 1300 1327 1924 96.5% chr17 - 23549785 23549812 28 browser details YourSeq 26 1299 1324 1924 100.0% chr14 - 25910342 25910367 26 browser details YourSeq 26 1299 1324 1924 100.0% chr14 - 26050092 26050117 26

Note: The 1924 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Ipo5 5 [ Mus musculus (house mouse) ] Gene ID: 70572, updated on 14-Aug-2019

Gene summary

Official Symbol Ipo5 provided by MGI Official Full Name importin 5 provided by MGI Primary source MGI:MGI:1917822 See related Ensembl:ENSMUSG00000030662 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 IMB3; imp5; Kpnb3; C76941; Ranbp5; AA409333; 1110011C18Rik; 5730478E03Rik Expression Broad expression in testis adult (RPKM 96.4), CNS E11.5 (RPKM 33.8) and 20 other tissues See more Orthologs human all

Genomic context

Location: 14; 14 E4-E5 See Ipo5 in Genome Data Viewer Exon count: 30

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (120896840..120948046)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (121310416..121347268)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 3 transcripts

Gene: Ipo5 ENSMUSG00000030662

Description importin 5 [Source:MGI Symbol;Acc:MGI:1917822] Gene Synonyms 1110011C18Rik, 5730478E03Rik, IMB3, Kpnb3, RanBP5, importin beta 3 Location Chromosome 14: 120,911,224-120,947,999 forward strand. GRCm38:CM001007.2 About this gene This gene has 3 transcripts (splice variants), 208 orthologues, 5 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ipo5-201 ENSMUST00000032898.8 4518 1097aa ENSMUSP00000032898.7 Protein coding CCDS37014 Q8BKC5 TSL:1 GENCODE basic APPRIS P1

Ipo5-202 ENSMUST00000227270.1 2094 No protein - Retained intron - - -

Ipo5-203 ENSMUST00000228277.1 4140 No protein - lncRNA - - -

56.78 kb Forward strand 120.91Mb 120.92Mb 120.93Mb 120.94Mb 120.95Mb Ipo5-201 >protein coding (Comprehensive set...

Ipo5-202 >retained intron

Ipo5-203 >lncRNA

Contigs < AC154283.1 Regulatory Build

120.91Mb 120.92Mb 120.93Mb 120.94Mb 120.95Mb Reverse strand 56.78 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000032898

36.78 kb Forward strand

Ipo5-201 >protein coding

ENSMUSP00000032... Low complexity (Seg) Superfamily Armadillo-type fold SMART TOG domain Pfam PF13646 HEAT repeat

Importin repeat 4 Importin repeat 6 PROSITE profiles Importin-beta, N-terminal domain PANTHER Importin beta family

PTHR10527:SF22

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 1097

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

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