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Mouse Tomm22 Knockout Project (CRISPR/Cas9)

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Mouse Tomm22 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Tomm22 Knockout Project (CRISPR/Cas9) Objective: To create a Tomm22 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tomm22 gene (NCBI Reference Sequence: NM_172609 ; Ensembl: ENSMUSG00000022427 ) is located on Mouse chromosome 15. 4 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 4 (Transcript: ENSMUST00000023062). Exon 1~4 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 1 starts from about 0.23% of the coding region. Exon 1~4 covers 100.0% of the coding region. The size of effective KO region: ~1397 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 Legends Exon of mouse Tomm22 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of start codon 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. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of stop codon 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(26.0% 520) | C(22.6% 452) | T(25.45% 509) | G(25.95% 519) Note: The 2000 bp section upstream of start codon 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(24.6% 492) | C(21.6% 432) | T(32.95% 659) | G(20.85% 417) Note: The 2000 bp section downstream of stop codon 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr15 + 79668893 79670892 2000 browser details YourSeq 345 1000 1723 2000 94.2% chr1 - 74416591 74811047 394457 browser details YourSeq 293 1004 1728 2000 92.6% chr1 - 49651627 50255281 603655 browser details YourSeq 268 1281 1729 2000 95.4% chr12 - 78949095 79108448 159354 browser details YourSeq 218 1451 1729 2000 94.0% chr14 + 76184044 76184386 343 browser details YourSeq 216 1447 1728 2000 93.0% chr2 - 160658068 160658629 562 browser details YourSeq 203 1068 1715 2000 88.0% chr1 - 161044323 161044786 464 browser details YourSeq 198 1076 1729 2000 87.7% chr10 + 116334627 116334980 354 browser details YourSeq 196 1069 1745 2000 86.0% chr10 + 117691682 117692121 440 browser details YourSeq 186 1068 1723 2000 94.8% chr5 + 140750930 140751587 658 browser details YourSeq 175 846 1173 2000 91.5% chr11 + 55385081 55385460 380 browser details YourSeq 170 1319 1729 2000 95.3% chr1 - 89062165 89062818 654 browser details YourSeq 166 1328 1737 2000 86.9% chr10 - 25337297 25337555 259 browser details YourSeq 161 1554 1729 2000 96.0% chr1 - 7745355 7745537 183 browser details YourSeq 161 1560 1744 2000 95.5% chr1 + 171429761 171429948 188 browser details YourSeq 160 1556 1729 2000 96.6% chr12 + 84842328 84842507 180 browser details YourSeq 159 1565 1744 2000 92.5% chr1 - 63244746 63244919 174 browser details YourSeq 156 1554 1729 2000 95.4% chr5 - 72244930 72245111 182 browser details YourSeq 156 1562 1729 2000 96.5% chr1 - 91518580 91518747 168 browser details YourSeq 156 1559 1728 2000 95.9% chr1 - 13169923 13170092 170 Note: The 2000 bp section upstream of start codon 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% chr15 + 79672290 79674289 2000 browser details YourSeq 542 1 578 2000 97.4% chr4 - 107759435 107760026 592 browser details YourSeq 531 1 578 2000 96.5% chr16 - 33137967 33138548 582 browser details YourSeq 385 13 578 2000 91.3% chr19 + 36818973 36819748 776 browser details YourSeq 275 140 576 2000 89.6% chr2 + 5284200 5284907 708 browser details YourSeq 263 13 569 2000 87.5% chr17 + 73448099 73448693 595 browser details YourSeq 130 765 1302 2000 91.3% chrX + 101279842 101574705 294864 browser details YourSeq 125 798 1344 2000 80.4% chr8 + 112035682 112035960 279 browser details YourSeq 122 787 1318 2000 93.0% chr1 - 16055842 16196340 140499 browser details YourSeq 121 404 575 2000 85.5% chr14 + 38198967 38199148 182 browser details YourSeq 120 732 1348 2000 80.0% chr19 - 4905393 4905808 416 browser details YourSeq 119 762 1376 2000 79.3% chr14 + 61594308 61594573 266 browser details YourSeq 115 749 1345 2000 77.5% chr12 - 106445528 106445686 159 browser details YourSeq 113 750 1349 2000 77.7% chr2 + 140583959 140584110 152 browser details YourSeq 112 1228 1371 2000 93.8% chr12 + 13334547 13334700 154 browser details YourSeq 111 747 1347 2000 76.9% chr5 + 40886679 40886838 160 browser details YourSeq 108 735 1340 2000 76.7% chrX - 23878024 23878212 189 browser details YourSeq 105 765 1343 2000 79.0% chr8 + 37640333 37640461 129 browser details YourSeq 104 1227 1349 2000 93.4% chr5 - 114293857 114293988 132 browser details YourSeq 104 1228 1349 2000 93.5% chr10 + 56455311 56455443 133 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Tomm22 translocase of outer mitochondrial membrane 22 [ Mus musculus (house mouse) ] Gene ID: 223696, updated on 10-Oct-2019 Gene summary Official Symbol Tomm22 provided by MGI Official Full Name translocase of outer mitochondrial membrane 22 provided by MGI Primary source MGI:MGI:2450248 See related Ensembl:ENSMUSG00000022427 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 Tom22; 2310047D01 Expression Ubiquitous expression in testis adult (RPKM 96.9), placenta adult (RPKM 50.2) and 28 other tissues See more Orthologs human all Genomic context Location: 15; 15 E1 See Tomm22 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (79670868..79672862) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (79501298..79503292) Chromosome 15 - NC_000081.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Tomm22 ENSMUSG00000022427 Description translocase of outer mitochondrial membrane 22 [Source:MGI Symbol;Acc:MGI:2450248] Gene Synonyms Tom22 Location Chromosome 15: 79,670,861-79,673,400 forward strand. GRCm38:CM001008.2 About this gene This gene has 3 transcripts (splice variants), 244 orthologues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tomm22- ENSMUST00000023062.4 1569 142aa ENSMUSP00000023062.3 Protein coding CCDS27646 Q9CPQ3 TSL:1 201 GENCODE basic APPRIS P2 Tomm22- ENSMUST00000127292.1 783 89aa ENSMUSP00000155226.1 Protein coding - A0A2R8VHM4 TSL:2 202 GENCODE basic APPRIS ALT2 Tomm22- ENSMUST00000229502.1 595 61aa ENSMUSP00000155148.1 Nonsense mediated - A0A2R8VHJ4 - 203 decay 22.54 kb Forward strand 79.665Mb 79.670Mb 79.675Mb 79.680Mb Genes (Comprehensive set... Cby1-201 >protein coding Tomm22-201 >protein coding Cby1-202 >retained intron Tomm22-203 >nonsense mediated decay Tomm22-202 >protein coding Contigs AC118227.12 > Genes < Josd1-201protein coding (Comprehensive set... < Gm49520-201processed pseudogene Regulatory Build 79.665Mb 79.670Mb 79.675Mb 79.680Mb Reverse strand 22.54 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000023062 2.54 kb Forward strand Tomm22-201 >protein coding ENSMUSP00000023... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Mitochondrial import receptor subunit Tom22 PANTHER Mitochondrial import receptor subunit Tom22 PTHR12504:SF3 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 142 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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