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Mouse Kpna4 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Kpna4 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Kpna4 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Kpna4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Kpna4 gene (NCBI Reference Sequence: NM_008467 ; Ensembl: ENSMUSG00000027782 ) is located on Mouse chromosome 3. 17 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 17 (Transcript: ENSMUST00000194558). Exon 6~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Kpna4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-323J1 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 6 starts from about 18.43% of the coding region. The knockout of Exon 6~7 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 5436 bp, and the size of intron 7 for 3'-loxP site insertion: 1756 bp. The size of effective cKO region: ~1463 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 6 7 8 17 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Kpna4 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com 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. 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7963bp) | A(30.73% 2447) | C(17.83% 1420) | T(32.24% 2567) | G(19.2% 1529) Note: The sequence of homologous arms and cKO region 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 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr3 - 69095866 69098865 3000 browser details YourSeq 156 1904 2097 3000 93.4% chr18 - 9675260 9675457 198 browser details YourSeq 152 1915 2109 3000 88.3% chr11 + 105231895 105232081 187 browser details YourSeq 149 1916 2104 3000 91.8% chr19 + 7005795 7005987 193 browser details YourSeq 148 1915 2103 3000 93.1% chr11 - 113772952 113773150 199 browser details YourSeq 147 1915 2096 3000 91.2% chr7 - 130478447 130478673 227 browser details YourSeq 146 1924 2104 3000 93.0% chr7 - 34148675 34148866 192 browser details YourSeq 146 1915 2103 3000 89.8% chr1 - 116741682 116741877 196 browser details YourSeq 145 1917 2121 3000 90.3% chr17 - 35537206 35537418 213 browser details YourSeq 145 1916 2103 3000 92.4% chr6 + 140687387 140687574 188 browser details YourSeq 144 1934 2103 3000 94.0% chr6 - 30583183 30583359 177 browser details YourSeq 144 1921 2104 3000 91.5% chr11 + 87419565 87419775 211 browser details YourSeq 143 1932 2104 3000 93.4% chr15 - 93515966 93516144 179 browser details YourSeq 141 1914 2118 3000 92.3% chr17 - 29498638 29498858 221 browser details YourSeq 141 1924 2103 3000 91.4% chrX + 105948537 105948898 362 browser details YourSeq 140 1924 2104 3000 91.7% chr7 - 16967760 16967950 191 browser details YourSeq 140 1930 2104 3000 92.3% chrX + 101510110 101510297 188 browser details YourSeq 140 1921 2101 3000 92.2% chr7 + 99547232 99547428 197 browser details YourSeq 140 1921 2103 3000 93.9% chr3 + 27219231 27219434 204 browser details YourSeq 139 1923 2098 3000 95.5% chr5 - 25700589 25700772 184 Note: The 3000 bp section upstream of Exon 6 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% chr3 - 69091403 69094402 3000 browser details YourSeq 199 405 2537 3000 91.7% chr13 - 61755111 62144617 389507 browser details YourSeq 194 409 2538 3000 90.9% chr18 + 36470223 36553089 82867 browser details YourSeq 140 405 565 3000 93.8% chr18 + 80206343 80206504 162 browser details YourSeq 139 404 565 3000 91.8% chr14 + 121765436 121765594 159 browser details YourSeq 139 405 564 3000 93.8% chr14 + 54353824 54353983 160 browser details YourSeq 137 405 566 3000 91.1% chr18 - 68797076 68797234 159 browser details YourSeq 137 405 563 3000 93.7% chr17 - 48462812 48462977 166 browser details YourSeq 136 2279 2557 3000 92.0% chr12 + 54800875 54801182 308 browser details YourSeq 135 405 558 3000 94.2% chr9 - 45837852 45838027 176 browser details YourSeq 135 406 567 3000 90.5% chr2 - 49831084 49831242 159 browser details YourSeq 135 406 566 3000 89.8% chr2 + 69711843 69711998 156 browser details YourSeq 134 405 568 3000 91.5% chr2 + 163628543 163628707 165 browser details YourSeq 133 405 558 3000 93.6% chr1 - 74447069 74447223 155 browser details YourSeq 132 405 550 3000 93.8% chr14 + 124712014 124712158 145 browser details YourSeq 131 409 560 3000 91.2% chr9 - 103225424 103225571 148 browser details YourSeq 131 405 564 3000 89.1% chr17 - 51946197 51946352 156 browser details YourSeq 129 405 549 3000 94.5% chr17 - 35055456 35055600 145 browser details YourSeq 129 404 549 3000 94.6% chr13 - 98830746 98830892 147 browser details YourSeq 128 409 566 3000 88.3% chr15 - 24182772 24182924 153 Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Kpna4 karyopherin (importin) alpha 4 [ Mus musculus (house mouse) ] Gene ID: 16649, updated on 10-Oct-2019 Gene summary Official Symbol Kpna4 provided by MGI Official Full Name karyopherin (importin) alpha 4 provided by MGI Primary source MGI:MGI:1100848 See related Ensembl:ENSMUSG00000027782 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as IPOA3; 1110058D08Rik Expression Ubiquitous expression in testis adult (RPKM 14.2), CNS E11.5 (RPKM 14.0) and 28 other tissues See more Orthologs human all Genomic context Location: 3; 3 E1 See Kpna4 in Genome Data Viewer Exon count: 17 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (69072221..69127092, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (68876143..68931014, complement) Chromosome 3 - NC_000069.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Kpna4 ENSMUSG00000027782 Description karyopherin (importin) alpha 4 [Source:MGI Symbol;Acc:MGI:1100848] Gene Synonyms 1110058D08Rik, IPOA3, importin alpha 3 Location Chromosome 3: 69,067,149-69,127,113 reverse strand. GRCm38:CM000996.2 About this gene This gene has 3 transcripts (splice variants), 204 orthologues, 6 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Kpna4-203 ENSMUST00000194558.5 3726 521aa ENSMUSP00000141227.1 Protein coding CCDS17403 O35343 Q4FJX1 TSL:1 GENCODE basic APPRIS P1 Kpna4-201 ENSMUST00000029353.8 8819 623aa ENSMUSP00000029353.3 Protein coding - A0A0B4J1E7 TSL:1 GENCODE basic Kpna4-202 ENSMUST00000127497.1 459 111aa ENSMUSP00000121076.1 Protein coding - D3YTN1 CDS 3' incomplete TSL:5 79.97 kb Forward strand 69.06Mb 69.08Mb 69.10Mb 69.12Mb Genes Gm1647-201 >lncRNA (Comprehensive set... Contigs AC119847.12 > Genes (Comprehensive set... < Kpna4-201protein coding < Kpna4-203protein coding < Gm22009-201scaRNA < Gm37558-201TEC < Kpna4-202protein coding Regulatory Build 69.06Mb 69.08Mb 69.10Mb 69.12Mb Reverse strand 79.97 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000194558 < Kpna4-203protein coding Reverse strand 54.87 kb ENSMUSP00000141... Low complexity (Seg) Superfamily Armadillo-type fold SMART Armadillo Pfam Importin-alpha, importin-beta-binding domain Atypical Arm repeat Armadillo PROSITE profiles Armadillo Importin-alpha, importin-beta-binding domain PIRSF Importin subunit alpha PANTHER PTHR23316:SF7 PTHR23316 Gene3D Armadillo-like helical Importin-alpha, importin-beta-binding domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 60 120 180 240 300 360 420 521 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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