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Mouse Tns4 Knockout Project (CRISPR/Cas9)

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Mouse Tns4 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Tns4 Knockout Project (CRISPR/Cas9) Objective: To create a Tns4 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tns4 gene (NCBI Reference Sequence: NM_172564 ; Ensembl: ENSMUSG00000017607 ) is located on Mouse chromosome 11. 13 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 13 (Transcript: ENSMUST00000017751). Exon 2~4 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 2 starts from the coding region. Exon 2~4 covers 58.81% of the coding region. The size of effective KO region: ~7781 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 13 Legends Exon of mouse Tns4 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 4 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. 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(22.35% 447) | C(28.25% 565) | T(26.55% 531) | G(22.85% 457) 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(28.6% 572) | C(22.5% 450) | T(23.8% 476) | G(25.1% 502) Note: The 2000 bp section downstream of Exon 4 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% chr11 - 99086094 99088093 2000 browser details YourSeq 41 426 499 2000 91.7% chr6 - 52135930 52136019 90 browser details YourSeq 39 394 446 2000 86.8% chr5 + 100795095 100795147 53 browser details YourSeq 38 423 499 2000 95.2% chr7 - 135079742 135079957 216 browser details YourSeq 37 418 476 2000 95.2% chr10 + 8947164 8947234 71 browser details YourSeq 35 418 499 2000 75.0% chr1 + 64364476 64364556 81 browser details YourSeq 33 396 446 2000 82.4% chr9 - 21206000 21206050 51 browser details YourSeq 33 449 493 2000 86.7% chr19 - 24057758 24057802 45 browser details YourSeq 32 404 440 2000 94.6% chr16 - 92743432 92743469 38 browser details YourSeq 32 455 499 2000 90.0% chr11 + 4156611 4156657 47 browser details YourSeq 31 443 493 2000 81.1% chr1 - 168099266 168099313 48 browser details YourSeq 31 393 435 2000 86.1% chr15 + 66978251 66978293 43 browser details YourSeq 31 449 493 2000 84.5% chr11 + 5497718 5497762 45 browser details YourSeq 29 396 444 2000 79.6% chr5 - 130851748 130851796 49 browser details YourSeq 29 425 457 2000 96.8% chr5 - 45359239 45359291 53 browser details YourSeq 29 425 458 2000 96.8% chr16 - 90173569 90173616 48 browser details YourSeq 29 463 498 2000 91.7% chr14 - 48587506 48587542 37 browser details YourSeq 29 425 457 2000 96.8% chr12 + 27471191 27471243 53 browser details YourSeq 28 470 499 2000 96.7% chr4 + 139596718 139596747 30 browser details YourSeq 28 425 456 2000 96.7% chr16 + 94807161 94807212 52 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% chr11 - 99076406 99078405 2000 browser details YourSeq 210 253 898 2000 81.5% chr4 + 116469956 116470363 408 browser details YourSeq 201 253 954 2000 82.9% chr2 + 163816191 163816755 565 browser details YourSeq 196 206 903 2000 81.7% chr11 - 59604336 59604761 426 browser details YourSeq 194 198 910 2000 82.1% chr14 - 75271853 75272343 491 browser details YourSeq 194 251 923 2000 81.7% chr5 + 148695196 148695545 350 browser details YourSeq 190 198 901 2000 81.4% chr7 + 127010446 127010851 406 browser details YourSeq 185 204 902 2000 90.2% chr5 - 28193066 28193896 831 browser details YourSeq 185 251 915 2000 82.5% chr4 - 120575910 120576292 383 browser details YourSeq 183 251 903 2000 81.5% chr16 + 30006939 30007287 349 browser details YourSeq 179 249 924 2000 75.2% chr7 + 128637724 128638121 398 browser details YourSeq 178 187 903 2000 77.6% chr13 + 54151448 54151927 480 browser details YourSeq 176 227 907 2000 84.5% chr9 - 44544108 44544717 610 browser details YourSeq 174 227 884 2000 79.2% chr2 - 4974469 4974856 388 browser details YourSeq 173 252 912 2000 81.6% chr13 - 43552194 43552584 391 browser details YourSeq 172 251 923 2000 82.0% chr1 + 171700232 171700721 490 browser details YourSeq 171 297 904 2000 83.5% chr9 + 103439933 103440296 364 browser details YourSeq 171 198 923 2000 80.9% chr4 + 58458355 58458843 489 browser details YourSeq 168 254 923 2000 78.5% chr11 + 121554245 121554659 415 browser details YourSeq 167 254 921 2000 84.2% chr2 - 128355701 128356115 415 Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Tns4 tensin 4 [ Mus musculus (house mouse) ] Gene ID: 217169, updated on 12-Aug-2019 Gene summary Official Symbol Tns4 provided by MGI Official Full Name tensin 4 provided by MGI Primary source MGI:MGI:2144377 See related Ensembl:ENSMUSG00000017607 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 Cten; AA589547; AU016405; 9930017A07Rik Expression Biased expression in colon adult (RPKM 41.6), large intestine adult (RPKM 12.5) and 6 other tissuesS ee more Orthologs human all Genomic context Location: 11; 11 D See Tns4 in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (99065678..99089306, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (98926992..98950620, complement) Chromosome 11 - NC_000077.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Tns4 ENSMUSG00000017607 Description tensin 4 [Source:MGI Symbol;Acc:MGI:2144377] Gene Synonyms 9930017A07Rik Location Chromosome 11: 99,065,678-99,089,306 reverse strand. GRCm38:CM001004.2 About this gene This gene has 4 transcripts (splice variants), 166 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tns4-201 ENSMUST00000017751.2 4755 696aa ENSMUSP00000017751.2 Protein coding CCDS25372 Q8BZ33 TSL:1 GENCODE basic APPRIS P1 Tns4-203 ENSMUST00000123303.1 763 No protein - Retained intron - - TSL:2 Tns4-204 ENSMUST00000153351.1 677 No protein - Retained intron - - TSL:5 Tns4-202 ENSMUST00000107465.7 678 No protein - lncRNA - - TSL:3 43.63 kb Forward strand 99.06Mb 99.07Mb 99.08Mb 99.09Mb Contigs AL591366.15 > Genes (Comprehensive set... < Tns4-201protein coding < Tns4-202lncRNA < Tns4-203retained intron < Tns4-204retained intron Regulatory Build 99.06Mb 99.07Mb 99.08Mb 99.09Mb Reverse strand 43.63 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000017751 < Tns4-201protein coding Reverse strand 23.63 kb ENSMUSP00000017... MobiDB lite Low complexity (Seg) Superfamily SH2 domain superfamily SSF50729 SMART SH2 domain PTB/PI domain Pfam SH2 domain Tensin/EPS8 phosphotyrosine-binding domain PROSITE profiles SH2 domain PANTHER PTHR45734 PTHR45734:SF6 Gene3D SH2 domain superfamily PH-like domain superfamily CDD Tensin, phosphotyrosine-binding domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 696 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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