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Mouse Ubiad1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ubiad1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ubiad1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ubiad1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ubiad1 gene (NCBI Reference Sequence: NM_027873 ; Ensembl: ENSMUSG00000047719 ) is located on Mouse chromosome 4. 2 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 2 (Transcript: ENSMUST00000051633). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ubiad1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-4F23 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 knock-out allele exhibit embryonic lethality, reduced embryo size, failure to form the primitive streak, no mesoderm and inability to synthesize menaquinone 4. Exon 1 covers 51.88% of the coding region. Start codon is in exon 1, and stop codon is in exon 2. The size of intron 1 for 3'-loxP site insertion: 7285 bp. The size of effective cKO region: ~783 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele A gRNA region T 5' G 3' 1 2 Targeting vector A T G Targeted allele A T G Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Ubiad1 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(7023bp) | A(26.73% 1877) | C(22.71% 1595) | T(25.17% 1768) | G(25.39% 1783) 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. 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% chr4 - 148444451 148447450 3000 browser details YourSeq 147 1773 2250 3000 87.6% chr11 + 97329429 97738489 409061 browser details YourSeq 135 1738 2294 3000 87.3% chr3 - 104536038 104536652 615 browser details YourSeq 118 1773 2263 3000 79.5% chr2 + 129797467 129797779 313 browser details YourSeq 115 582 972 3000 82.0% chr8 + 83155093 83155428 336 browser details YourSeq 110 2134 2292 3000 85.3% chr16 - 65776101 65776243 143 browser details YourSeq 109 582 971 3000 82.4% chr1 + 110900042 110900348 307 browser details YourSeq 108 2139 2294 3000 85.9% chr9 + 117315864 117316007 144 browser details YourSeq 104 2140 2298 3000 88.3% chr9 - 61285447 61285598 152 browser details YourSeq 103 2139 2276 3000 88.2% chr10 - 59867995 59868126 132 browser details YourSeq 98 564 965 3000 78.3% chr1 - 75178329 75178623 295 browser details YourSeq 97 2134 2287 3000 92.7% chr9 - 64136641 64136793 153 browser details YourSeq 97 1694 2252 3000 76.3% chr12 - 17180885 17181219 335 browser details YourSeq 96 2135 2263 3000 88.1% chrX - 73750195 73750317 123 browser details YourSeq 96 1543 2250 3000 74.2% chrX + 101076636 101076889 254 browser details YourSeq 95 2140 2292 3000 91.2% chr11 + 120881460 120881613 154 browser details YourSeq 94 2139 2263 3000 89.6% chr4 + 115834999 115835118 120 browser details YourSeq 93 2139 2263 3000 88.5% chrX - 10328310 10328428 119 browser details YourSeq 93 2134 2250 3000 92.0% chr3 - 96873074 96873187 114 browser details YourSeq 92 1667 1845 3000 93.6% chr3 + 65118227 65515476 397250 Note: The 3000 bp section upstream of Exon 1 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% chr4 - 148440678 148443677 3000 browser details YourSeq 368 28 2939 3000 93.0% chr7 + 16636262 16779966 143705 browser details YourSeq 235 17 644 3000 95.1% chr4 - 120596586 120877229 280644 browser details YourSeq 224 17 2963 3000 92.9% chr3 - 51280499 51317743 37245 browser details YourSeq 214 29 618 3000 87.8% chr18 - 61343139 61343444 306 browser details YourSeq 213 26 623 3000 85.6% chr4 - 137674337 137674644 308 browser details YourSeq 212 17 616 3000 87.1% chr2 + 60798317 60798638 322 browser details YourSeq 210 29 830 3000 85.7% chr17 + 47510175 47510511 337 browser details YourSeq 187 53 623 3000 94.4% chr13 + 93593768 93594398 631 browser details YourSeq 183 57 623 3000 93.4% chr3 + 58579802 58580428 627 browser details YourSeq 177 17 629 3000 82.9% chr16 + 32110784 32111092 309 browser details YourSeq 173 46 616 3000 85.0% chr14 - 25993120 25993391 272 browser details YourSeq 168 22 650 3000 86.2% chr5 + 100824862 100825285 424 browser details YourSeq 165 69 612 3000 89.1% chr2 - 156005761 156006266 506 browser details YourSeq 158 465 645 3000 91.9% chr12 + 76516776 76516948 173 browser details YourSeq 155 41 612 3000 82.7% chr2 + 30420077 30420367 291 browser details YourSeq 154 471 830 3000 84.5% chr10 + 75849821 75849994 174 browser details YourSeq 151 17 612 3000 84.7% chr10 - 67114548 67114894 347 browser details YourSeq 148 503 749 3000 92.4% chr9 - 65246110 65246355 246 browser details YourSeq 148 466 830 3000 84.5% chr1 + 161093291 161093467 177 Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ubiad1 UbiA prenyltransferase domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 71707, updated on 21-Oct-2019 Gene summary Official Symbol Ubiad1 provided by MGI Official Full Name UbiA prenyltransferase domain containing 1 provided by MGI Primary source MGI:MGI:1918957 See related Ensembl:ENSMUSG00000047719 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 Tere1; AI426463; AW320947; 1200002M06Rik Expression Ubiquitous expression in kidney adult (RPKM 20.7), ovary adult (RPKM 13.0) and 28 other tissues See more Orthologs human all Genomic context Location: 4; 4 E2 See Ubiad1 in Genome Data Viewer Exon count: 2 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (148434495..148444760, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (147808606..147818860, complement) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Ubiad1 ENSMUSG00000047719 Description UbiA prenyltransferase domain containing 1 [Source:MGI Symbol;Acc:MGI:1918957] Gene Synonyms 1200002M06Rik, Tere1 Location Chromosome 4: 148,434,495-148,444,771 reverse strand. GRCm38:CM000997.2 About this gene This gene has 2 transcripts (splice variants), 187 orthologues, is a member of 1 Ensembl protein family and is associated with 16 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ubiad1-201 ENSMUST00000051633.2 2992 336aa ENSMUSP00000058502.2 Protein coding CCDS18936 Q9DC60 TSL:1 GENCODE basic APPRIS P1 Ubiad1-202 ENSMUST00000156686.1 576 No protein - lncRNA - - TSL:2 30.28 kb Forward strand 148.43Mb 148.44Mb 148.45Mb Genes Mtor-202 >protein coding (Comprehensive set... Mtor-203 >retained intron Mtor-201 >protein coding Contigs AL606919.12 > AL731654.7 > Genes (Comprehensive set... < Ubiad1-201protein coding < Ubiad1-202lncRNA < Gm23303-201snRNA Regulatory Build 148.43Mb 148.44Mb 148.45Mb Reverse strand 30.28 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000051633 < Ubiad1-201protein coding Reverse strand 10.28 kb ENSMUSP00000058... Transmembrane heli... Low complexity (Seg) Pfam UbiA prenyltransferase family PIRSF UbiA prenyltransferase domain containing protein 1 PANTHER PTHR13929:SF0 PTHR13929 CDD cd13962 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 336 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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