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Mouse Tc2n Knockout Project (CRISPR/Cas9)

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Mouse Tc2n Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Tc2n Knockout Project (CRISPR/Cas9) Objective: To create a Tc2n knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tc2n gene (NCBI Reference Sequence: NM_001082976 ; Ensembl: ENSMUSG00000021187 ) is located on Mouse chromosome 12. 12 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000162735). Exon 2~3 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 the coding region. Exon 2~3 covers 20.31% of the coding region. The size of effective KO region: ~3154 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 12 Legends Exon of mouse Tc2n 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 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 2000 bp section downstream of Exon 3 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(31.5% 630) | C(15.95% 319) | T(34.9% 698) | G(17.65% 353) 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(2000bp) | A(30.6% 612) | C(14.2% 284) | T(40.2% 804) | G(15.0% 300) Note: The 2000 bp section downstream of Exon 3 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% chr12 - 101709491 101711490 2000 browser details YourSeq 177 519 711 2000 95.9% chr17 - 37137492 37137684 193 browser details YourSeq 148 1 884 2000 91.3% chr18 + 6402100 7055232 653133 browser details YourSeq 112 149 392 2000 84.0% chr1 - 136400173 136400763 591 browser details YourSeq 98 780 1072 2000 90.9% chr7 - 59189877 59190402 526 browser details YourSeq 98 764 884 2000 91.7% chr14 - 105594808 105594936 129 browser details YourSeq 97 765 881 2000 92.4% chr3 - 50458588 50458711 124 browser details YourSeq 96 762 884 2000 89.5% chr4 + 25323479 25323605 127 browser details YourSeq 94 168 388 2000 81.0% chr7 + 116751985 116752207 223 browser details YourSeq 94 775 884 2000 93.7% chr2 + 85218875 85218996 122 browser details YourSeq 92 701 860 2000 91.2% chr5 - 45326213 45326548 336 browser details YourSeq 92 768 884 2000 89.8% chr15 - 92932824 92932944 121 browser details YourSeq 92 605 868 2000 92.7% chr14 + 94827616 94828006 391 browser details YourSeq 91 768 884 2000 88.9% chr14 - 124582274 124582390 117 browser details YourSeq 91 764 884 2000 91.0% chr7 + 114341922 114342058 137 browser details YourSeq 90 764 884 2000 85.6% chr13 + 89019468 89019586 119 browser details YourSeq 89 781 883 2000 93.3% chrX - 98733052 98733154 103 browser details YourSeq 89 768 875 2000 91.6% chrX - 43716688 43716798 111 browser details YourSeq 89 114 386 2000 89.3% chr14 - 34784120 34784436 317 browser details YourSeq 89 781 884 2000 93.3% chr14 - 9915399 9915507 109 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 2000 1 2000 2000 100.0% chr12 - 101704383 101706382 2000 browser details YourSeq 71 227 383 2000 82.0% chr5 - 105654086 105654213 128 browser details YourSeq 68 236 410 2000 91.4% chr16 - 8376589 8376774 186 browser details YourSeq 66 267 387 2000 78.8% chr4 + 139077341 139077439 99 browser details YourSeq 64 340 442 2000 76.6% chr8 + 47407495 47407592 98 browser details YourSeq 62 302 417 2000 88.8% chr4 - 70481373 70481488 116 browser details YourSeq 62 236 430 2000 73.4% chr19 - 57120314 57120482 169 browser details YourSeq 58 299 410 2000 92.7% chr5 + 75192063 75192174 112 browser details YourSeq 55 338 420 2000 91.0% chr4 - 138472620 138472703 84 browser details YourSeq 53 294 390 2000 93.5% chr7 - 120683818 120683914 97 browser details YourSeq 53 331 404 2000 86.5% chr4 - 129434345 129434419 75 browser details YourSeq 53 301 411 2000 89.6% chr7 + 35884295 35884405 111 browser details YourSeq 52 340 409 2000 85.3% chr2 + 6108537 6108605 69 browser details YourSeq 52 267 407 2000 84.5% chr17 + 34388668 34388802 135 browser details YourSeq 51 296 387 2000 80.0% chr18 - 53086690 53086779 90 browser details YourSeq 51 324 395 2000 89.4% chr11 - 72543569 72543642 74 browser details YourSeq 50 333 404 2000 86.0% chr4 + 106990425 106990495 71 browser details YourSeq 49 350 416 2000 84.7% chr7 - 112214880 112214945 66 browser details YourSeq 49 296 395 2000 87.8% chr11 - 76690980 76691077 98 browser details YourSeq 49 296 420 2000 83.1% chr5 + 149959304 149959426 123 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Tc2n tandem C2 domains, nuclear [ Mus musculus (house mouse) ] Gene ID: 74413, updated on 12-Aug-2019 Gene summary Official Symbol Tc2n provided by MGI Official Full Name tandem C2 domains, nuclear provided by MGI Primary source MGI:MGI:1921663 See related Ensembl:ENSMUSG00000021187 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 Tac2-N; Mtac2d1; 4933406D09Rik Expression Biased expression in bladder adult (RPKM 2.4), testis adult (RPKM 0.5) and 6 other tissues See more Orthologs human all Genomic context Location: 12; 12 E See Tc2n in Genome Data Viewer Exon count: 18 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (101645446..101718919, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (102886812..102956712, complement) Chromosome 12 - NC_000078.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Tc2n ENSMUSG00000021187 Description tandem C2 domains, nuclear [Source:MGI Symbol;Acc:MGI:1921663] Gene Synonyms 4933406D09Rik, Mtac2d1, Tac2-N Location Chromosome 12: 101,645,443-101,718,523 reverse strand. GRCm38:CM001005.2 About this gene This gene has 4 transcripts (splice variants), 193 orthologues, 31 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tc2n-204 ENSMUST00000162735.7 5290 489aa ENSMUSP00000125288.1 Protein coding CCDS36524 Q91XT6 TSL:1 GENCODE basic APPRIS P1 Tc2n-203 ENSMUST00000160830.1 2005 489aa ENSMUSP00000124287.1 Protein coding CCDS36524 Q91XT6 TSL:1 GENCODE basic APPRIS P1 Tc2n-202 ENSMUST00000160715.7 1386 425aa ENSMUSP00000125099.1 Protein coding - E0CXQ6 TSL:5 GENCODE basic Tc2n-201 ENSMUST00000110047.8 2587 489aa ENSMUSP00000105674.2 Nonsense mediated decay - Q91XT6 TSL:2 93.08 kb Forward strand 101.64Mb 101.66Mb 101.68Mb 101.70Mb 101.72Mb Genes Gm16285-201 >processed pseudogene (Comprehensive set... Contigs AC125355.4 > AC122473.4 > Genes (Comprehensive set... < Tc2n-204protein coding < Tc2n-201nonsense mediated decay < Tc2n-202protein coding < Tc2n-203protein coding Regulatory Build 101.64Mb 101.66Mb 101.68Mb 101.70Mb 101.72Mb Reverse strand 93.08 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript pseudogene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000162735 < Tc2n-204protein coding Reverse strand 73.08 kb ENSMUSP00000125... MobiDB lite Low complexity (Seg) Superfamily SSF49562 SMART C2 domain Pfam C2 domain PROSITE profiles C2 domain PANTHER Tandem C2 domains nuclear protein Gene3D C2 domain superfamily CDD Tandem C2 domains nuclear protein, C2A domain Tandem C2 domains nuclear protein, C2B domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 489 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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