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Mouse Ttyh2 Knockout Project (CRISPR/Cas9)

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Mouse Ttyh2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ttyh2 Knockout Project (CRISPR/Cas9) Objective: To create a Ttyh2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ttyh2 gene (NCBI Reference Sequence: NM_053273 ; Ensembl: ENSMUSG00000034714 ) is located on Mouse chromosome 11. 14 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 14 (Transcript: ENSMUST00000045779). Exon 2 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 about 8.15% of the coding region. Exon 2 covers 10.84% of the coding region. The size of effective KO region: ~173 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 14 Legends Exon of mouse Ttyh2 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. 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 Exon 2 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(22.25% 445) | C(30.75% 615) | T(22.2% 444) | G(24.8% 496) 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(25.85% 517) | C(21.15% 423) | T(23.85% 477) | G(29.15% 583) Note: The 2000 bp section downstream 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. 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 + 114684395 114686394 2000 browser details YourSeq 64 240 318 2000 95.7% chr15 + 102764838 102765073 236 browser details YourSeq 63 27 300 2000 76.9% chr2 - 154905806 154906010 205 browser details YourSeq 60 238 300 2000 98.5% chr15 - 6597473 6597537 65 browser details YourSeq 59 236 300 2000 96.9% chr16 - 84549630 84549699 70 browser details YourSeq 59 232 300 2000 87.9% chr6 + 7628926 7628991 66 browser details YourSeq 58 233 300 2000 87.7% chr5 - 126379527 126379591 65 browser details YourSeq 58 234 300 2000 94.1% chr16 + 9006507 9006575 69 browser details YourSeq 57 238 300 2000 90.0% chr7 + 78614947 78615006 60 browser details YourSeq 56 239 300 2000 96.7% chr8 + 124572485 124572552 68 browser details YourSeq 56 959 1014 2000 100.0% chr19 + 15167088 15167143 56 browser details YourSeq 55 238 300 2000 88.4% chr18 - 26751868 26751927 60 browser details YourSeq 55 238 300 2000 88.4% chr14 - 22003556 22003615 60 browser details YourSeq 55 238 300 2000 88.4% chr3 + 152279766 152279825 60 browser details YourSeq 55 239 300 2000 95.1% chr11 + 30954145 30954209 65 browser details YourSeq 55 236 300 2000 88.6% chr10 + 14994076 14994137 62 browser details YourSeq 55 236 298 2000 95.3% chr1 + 159058697 159058965 269 browser details YourSeq 54 238 300 2000 95.0% chrX - 168409461 168409526 66 browser details YourSeq 54 238 297 2000 89.5% chr9 - 25900347 25900403 57 browser details YourSeq 54 238 298 2000 96.7% chr4 + 8642230 8642304 75 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 + 114686568 114688567 2000 browser details YourSeq 149 1395 2000 2000 87.8% chr7 + 101956875 101957462 588 browser details YourSeq 128 1434 1997 2000 85.6% chr16 + 11238743 11239263 521 browser details YourSeq 125 1861 2000 2000 95.0% chr8 - 91179898 91180037 140 browser details YourSeq 122 1866 2000 2000 93.3% chr10 + 117713896 117714028 133 browser details YourSeq 120 1863 2000 2000 91.7% chr5 - 102731303 102731435 133 browser details YourSeq 120 1867 2000 2000 95.5% chr4 - 109810428 109810562 135 browser details YourSeq 119 1866 2000 2000 92.5% chr18 - 54083740 54083872 133 browser details YourSeq 119 1866 2000 2000 95.5% chr14 - 74838345 74838483 139 browser details YourSeq 119 1867 2000 2000 95.5% chr13 - 93875935 93876184 250 browser details YourSeq 118 1863 2000 2000 92.0% chr4 - 108279229 108279365 137 browser details YourSeq 118 1868 2000 2000 94.8% chr12 + 30920439 30920572 134 browser details YourSeq 118 1866 2000 2000 92.5% chr1 + 157504935 157505067 133 browser details YourSeq 117 1866 2000 2000 94.1% chr5 - 120446339 120446478 140 browser details YourSeq 117 1866 2000 2000 94.0% chr2 - 58794049 58794186 138 browser details YourSeq 117 1866 2000 2000 94.7% chr16 - 37739810 37739944 135 browser details YourSeq 117 1866 2000 2000 94.1% chr4 + 100060345 100060480 136 browser details YourSeq 116 1869 2000 2000 94.7% chrX - 92491694 92491827 134 browser details YourSeq 116 1865 2000 2000 94.0% chr5 - 109667590 109667728 139 browser details YourSeq 116 1866 2000 2000 96.0% chr2 - 33524889 33525022 134 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Ttyh2 tweety family member 2 [ Mus musculus (house mouse) ] Gene ID: 117160, updated on 12-Aug-2019 Gene summary Official Symbol Ttyh2 provided by MGI Official Full Name tweety family member 2 provided by MGI Primary source MGI:MGI:2157091 See related Ensembl:ENSMUSG00000034714 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 1110001A03Rik Expression Broad expression in adrenal adult (RPKM 72.1), small intestine adult (RPKM 41.6) and 22 other tissues See more Orthologs human all Genomic context Location: 11; 11 E2 See Ttyh2 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (114675468..114720984) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (114536782..114582298) Chromosome 11 - NC_000077.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Ttyh2 ENSMUSG00000034714 Description tweety family member 2 [Source:MGI Symbol;Acc:MGI:2157091] Gene Synonyms 1110001A03Rik Location Chromosome 11: 114,675,431-114,720,977 forward strand. GRCm38:CM001004.2 About this gene This gene has 2 transcripts (splice variants), 250 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ttyh2-201 ENSMUST00000045779.5 3486 532aa ENSMUSP00000037821.5 Protein coding CCDS25608 Q3TH73 TSL:1 GENCODE basic APPRIS P1 Ttyh2-202 ENSMUST00000141111.1 695 No protein - lncRNA - - TSL:2 65.55 kb Forward strand 114.68Mb 114.70Mb 114.72Mb Genes Rpl38-204 >protein coding Dnaic2-201 >protein coding (Comprehensive set... Rpl38-201 >protein coding Dnaic2-202 >protein coding Rpl38-203 >protein coding Dnaic2-207 >lncRNA Rpl38-202 >protein coding Dnaic2-204 >lncRNA Ttyh2-201 >protein coding Ttyh2-202 >lncRNA Contigs AL645484.14 > AL663079.9 > Regulatory Build 114.68Mb 114.70Mb 114.72Mb Reverse strand 65.55 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 RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000045779 45.55 kb Forward strand Ttyh2-201 >protein coding ENSMUSP00000037... Transmembrane heli... Low complexity (Seg) Pfam Tweety PANTHER Tweety PTHR12424:SF6 CDD Tweety All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 532 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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