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Mouse Vipas39 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Vipas39 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Vipas39 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Vipas39 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Vipas39 gene (NCBI Reference Sequence: NM_001142580 ; Ensembl: ENSMUSG00000021038 ) is located on Mouse chromosome 12. 20 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 20 (Transcript: ENSMUST00000072744). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Vipas39 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-213E8 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: Mice homozygous for a conditional allele activated by an inducible cre exhibit dry and scaly skin, hair loss, and defects in tail tendon collagen I structure. Exon 3 starts from about 6.38% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 3170 bp, and the size of intron 3 for 3'-loxP site insertion: 1410 bp. The size of effective cKO region: ~603 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 3 4 20 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Vipas39 Homology arm cKO region loxP site Page 2 of 8 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(7103bp) | A(24.96% 1773) | C(21.12% 1500) | T(31.56% 2242) | G(22.36% 1588) 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 8 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% chr12 - 87260985 87263984 3000 browser details YourSeq 274 1738 2302 3000 88.5% chr10 + 120338059 120338781 723 browser details YourSeq 263 1702 2302 3000 87.9% chr7 + 67544354 67545008 655 browser details YourSeq 235 1738 2302 3000 88.2% chr5 + 77141929 77142645 717 browser details YourSeq 231 1738 2302 3000 84.7% chr10 + 76128743 76129275 533 browser details YourSeq 215 2021 2389 3000 87.5% chr5 + 101904760 101905495 736 browser details YourSeq 211 1967 2352 3000 83.7% chr5 - 35309216 35309677 462 browser details YourSeq 210 2039 2387 3000 82.8% chr10 + 116978203 116978530 328 browser details YourSeq 206 1750 2302 3000 82.2% chr15 - 98361868 98362261 394 browser details YourSeq 203 2039 2357 3000 86.8% chr15 + 96579589 96579894 306 browser details YourSeq 200 2039 2352 3000 85.8% chr15 + 88484785 88500090 15306 browser details YourSeq 197 2049 2788 3000 80.3% chr1 + 83343815 83344204 390 browser details YourSeq 194 2039 2350 3000 83.4% chr3 - 34716512 34716819 308 browser details YourSeq 193 2039 2389 3000 86.3% chr10 + 60633169 60633510 342 browser details YourSeq 190 2039 2349 3000 85.5% chr10 - 69142259 69142564 306 browser details YourSeq 187 2039 2302 3000 86.4% chr13 - 51782872 51783137 266 browser details YourSeq 187 2145 2758 3000 83.5% chr16 + 84947085 84947417 333 browser details YourSeq 186 2042 2352 3000 84.1% chr16 + 14091939 14092246 308 browser details YourSeq 183 2039 2302 3000 85.2% chr13 - 53940571 54038600 98030 browser details YourSeq 183 2050 2743 3000 84.4% chr6 + 148300987 148301470 484 Note: The 3000 bp section upstream of Exon 3 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% chr12 - 87257382 87260381 3000 browser details YourSeq 307 248 627 3000 94.0% chr4 + 116680833 116681247 415 browser details YourSeq 306 252 607 3000 95.3% chr5 - 23823657 23824015 359 browser details YourSeq 305 276 778 3000 90.1% chr2 - 32025645 32102217 76573 browser details YourSeq 302 248 609 3000 94.7% chr19 + 8850010 8865447 15438 browser details YourSeq 297 248 609 3000 93.1% chr1 + 164024543 164025082 540 browser details YourSeq 296 248 609 3000 94.4% chr7 - 12999955 13000323 369 browser details YourSeq 292 271 640 3000 93.3% chr10 + 128222946 128223671 726 browser details YourSeq 288 265 609 3000 94.2% chr11 - 6609580 6609966 387 browser details YourSeq 288 293 611 3000 98.1% chr2 + 130670444 130779345 108902 browser details YourSeq 284 252 608 3000 94.2% chr15 + 8510639 8511181 543 browser details YourSeq 283 283 637 3000 93.3% chr4 + 129296502 129297154 653 browser details YourSeq 281 267 628 3000 91.6% chr17 + 5276335 5276681 347 browser details YourSeq 278 287 609 3000 95.1% chr17 - 83612280 83612601 322 browser details YourSeq 272 258 607 3000 92.9% chr1 + 131157684 131158028 345 browser details YourSeq 253 314 608 3000 96.7% chr2 - 5843581 5844246 666 browser details YourSeq 249 266 608 3000 95.3% chr6 + 38585429 38585826 398 browser details YourSeq 243 297 608 3000 97.0% chr17 - 32261386 32261708 323 browser details YourSeq 234 419 799 3000 93.1% chr12 + 91797646 91798268 623 browser details YourSeq 223 263 607 3000 89.6% chr2 - 59124708 59124974 267 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Vipas39 VPS33B interacting protein, apical-basolateral polarity regulator, spe-39 homolog [ Mus musculus (house mouse) ] Gene ID: 104799, updated on 12-Aug-2019 Gene summary Official Symbol Vipas39 provided by MGI Official Full Name VPS33B interacting protein, apical-basolateral polarity regulator, spe-39 homolog provided by MGI Primary source MGI:MGI:2144805 See related Ensembl:ENSMUSG00000021038 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 Spe39; Vipar; SPE-39; hSPE-39; AI413782; 6720456H09Rik; 9330175H22Rik Expression Ubiquitous expression in CNS E14 (RPKM 16.2), CNS E18 (RPKM 15.9) and 28 other tissues See more Orthologs human all Genomic context Location: 12; 12 D2 See Vipas39 in Genome Data Viewer Exon count: 22 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (87238875..87266378, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (88579825..88607236, complement) Chromosome 12 - NC_000078.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Vipas39 ENSMUSG00000021038 Description VPS33B interacting protein, apical-basolateral polarity regulator, spe-39 homolog [Source:MGI Symbol;Acc:MGI:2144805] Gene Synonyms SPE-39, Vipar Location Chromosome 12: 87,238,868-87,266,256 reverse strand. GRCm38:CM001005.2 About this gene This gene has 6 transcripts (splice variants), 203 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Vipas39-202 ENSMUST00000072744.14 2524 491aa ENSMUSP00000072527.7 Protein coding CCDS49119 Q8BGQ1 TSL:1 GENCODE basic APPRIS P1 Vipas39-201 ENSMUST00000021426.9 2509 472aa ENSMUSP00000021426.9 Protein coding CCDS26074 Q8BGQ1 TSL:1 GENCODE basic Vipas39-203 ENSMUST00000179379.8 2418 472aa ENSMUSP00000137190.1 Protein coding CCDS26074 Q8BGQ1 TSL:1 GENCODE basic Vipas39-206 ENSMUST00000222350.1 1790 No protein - Retained intron - - TSL:1 Vipas39-204 ENSMUST00000220858.1 1398 No protein - Retained intron - - TSL:2 Vipas39-205 ENSMUST00000221707.1 417 No protein - Retained intron - - TSL:3 Page 6 of 8 https://www.alphaknockout.com 47.39 kb Forward strand 87.23Mb 87.24Mb 87.25Mb 87.26Mb 87.27Mb Genes Ahsa1-205 >protein coding (Comprehensive set... Ahsa1-201 >protein coding Ahsa1-202 >retained intron Gm47293-201 >miRNA Ahsa1-203 >retained intron Ahsa1-204 >retained intron Contigs AC151967.6 > Genes (Comprehensive set... < Noxred1-203protein coding < Vipas39-204retained intro<n Vipas39-205retained intron < Noxred1-201protein coding < Vipas39-202protein coding < Noxred1-202protein coding < Vipas39-203protein coding < Vipas39-201protein coding < Vipas39-206retained intron Regulatory Build 87.23Mb 87.24Mb 87.25Mb 87.26Mb 87.27Mb Reverse strand 47.39 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000072744 < Vipas39-202protein coding Reverse strand 27.38 kb ENSMUSP00000072... MobiDB lite Low complexity (Seg) Pfam Vps16, C-terminal PANTHER PTHR13364:SF6 Spermatogenesis-defective protein 39 Gene3D Vps16, C-terminal domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 491 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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