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Mouse Pou2f3 Knockout Project (CRISPR/Cas9)

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Mouse Pou2f3 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pou2f3 Knockout Project (CRISPR/Cas9) Objective: To create a Pou2f3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pou2f3 gene (NCBI Reference Sequence: NM_011139 ; Ensembl: ENSMUSG00000032015 ) is located on Mouse chromosome 9. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000176636). Exon 4~8 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: Mice homozygous for one null mutation exhibit defective keratinocyte differentiation, however the skin and coat appear normal. Mice homozygous for another null mutation display loss of sweet, umami and bitter taste perception and expansion of sour taste receptor cells. Exon 4 starts from about 10.29% of the coding region. Exon 4~8 covers 47.64% of the coding region. The size of effective KO region: ~7599 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 4 5 6 7 8 13 Legends Exon of mouse Pou2f3 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 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. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1884 bp section downstream of Exon 8 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(19.5% 390) | C(25.2% 504) | T(34.35% 687) | G(20.95% 419) Note: The 2000 bp section upstream 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(1884bp) | A(25.16% 474) | C(23.25% 438) | T(24.63% 464) | G(26.96% 508) Note: The 1884 bp section downstream of Exon 8 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% chr9 - 43146915 43148914 2000 browser details YourSeq 262 1380 1719 2000 92.9% chr18 - 44548046 44548491 446 browser details YourSeq 230 1397 1725 2000 95.3% chr18 + 40242561 40242937 377 browser details YourSeq 222 1150 1716 2000 94.8% chr11 - 84163622 84164220 599 browser details YourSeq 217 1412 1724 2000 94.3% chr7 - 89071865 89072222 358 browser details YourSeq 194 1482 1716 2000 91.9% chr9 - 20780489 20780700 212 browser details YourSeq 175 1520 1720 2000 93.7% chrX - 111593938 111594134 197 browser details YourSeq 174 1448 1724 2000 93.1% chr14 - 86269856 86270299 444 browser details YourSeq 171 1188 1739 2000 94.8% chr5 - 120308616 120309180 565 browser details YourSeq 165 1241 1724 2000 93.7% chr13 - 64217429 64217984 556 browser details YourSeq 164 1467 1733 2000 87.1% chr1 + 133062678 133062889 212 browser details YourSeq 162 1393 1716 2000 87.3% chr2 + 156548736 156548918 183 browser details YourSeq 161 1444 1726 2000 87.0% chr4 - 53105377 53105552 176 browser details YourSeq 160 1559 1740 2000 95.0% chr11 - 97304287 97304491 205 browser details YourSeq 160 1433 1724 2000 84.3% chr5 + 107349298 107349506 209 browser details YourSeq 160 1304 1716 2000 87.1% chr2 + 119695040 119695293 254 browser details YourSeq 158 1458 1716 2000 92.5% chr11 - 78809214 78809466 253 browser details YourSeq 158 1546 1732 2000 94.0% chr7 + 127067874 127068093 220 browser details YourSeq 156 1559 1734 2000 92.4% chr11 + 105608672 105608843 172 browser details YourSeq 153 1555 1739 2000 93.3% chrX - 66630730 66630926 197 Note: The 2000 bp section upstream of Exon 4 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 1884 1 1884 1884 100.0% chr9 - 43137432 43139315 1884 browser details YourSeq 270 1443 1765 1884 92.5% chr10 + 120039287 120039636 350 browser details YourSeq 261 1445 1765 1884 92.9% chr8 + 118030153 118030475 323 browser details YourSeq 258 1443 1765 1884 90.5% chr13 - 58019601 58019926 326 browser details YourSeq 258 1442 1765 1884 92.9% chr2 + 72844770 72845095 326 browser details YourSeq 256 1443 1764 1884 90.1% chr6 - 18480829 18481152 324 browser details YourSeq 256 1437 1765 1884 91.2% chr14 + 35128838 35129190 353 browser details YourSeq 254 1441 1739 1884 93.0% chr12 - 111399672 111492352 92681 browser details YourSeq 253 1434 1765 1884 87.6% chr9 - 83534152 83534481 330 browser details YourSeq 253 1442 1765 1884 90.2% chr18 - 53404997 53405322 326 browser details YourSeq 252 1442 1750 1884 91.0% chr3 - 146480241 146480550 310 browser details YourSeq 252 1446 1765 1884 91.0% chr9 + 45214721 45215063 343 browser details YourSeq 251 1442 1742 1884 92.1% chrX - 94336584 94336886 303 browser details YourSeq 251 1441 1765 1884 91.5% chr14 + 65273024 65273371 348 browser details YourSeq 250 1451 1765 1884 92.1% chr1 - 157108869 157109185 317 browser details YourSeq 250 1443 1765 1884 88.9% chr19 + 24286247 24286570 324 browser details YourSeq 250 1451 1824 1884 89.2% chr16 + 73055115 73055456 342 browser details YourSeq 250 1444 1762 1884 89.6% chr14 + 118829985 118830303 319 browser details YourSeq 249 1442 1765 1884 88.6% chr10 - 11431233 11431557 325 browser details YourSeq 248 1443 1765 1884 88.8% chr11 - 114912600 114912922 323 Note: The 1884 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Pou2f3 POU domain, class 2, transcription factor 3 [ Mus musculus (house mouse) ] Gene ID: 18988, updated on 10-Oct-2019 Gene summary Official Symbol Pou2f3 provided by MGI Official Full Name POU domain, class 2, transcription factor 3 provided by MGI Primary source MGI:MGI:102565 See related Ensembl:ENSMUSG00000032015 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 Skin; Oct11; Otf11; Epoc-1; Otf-11; Skn-1a; Skn-li; Oct-11a; Skin-1a Expression Biased expression in limb E14.5 (RPKM 2.0), placenta adult (RPKM 1.8) and 11 other tissues See more Orthologs human all Genomic context Location: 9 A5.1; 9 24.21 cM See Pou2f3 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (43123927..43205755, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (42932022..43013838, complement) Chromosome 9 - NC_000075.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Pou2f3 ENSMUSG00000032015 Description POU domain, class 2, transcription factor 3 [Source:MGI Symbol;Acc:MGI:102565] Gene Synonyms Epoc-1, Oct-11a, Oct11, Otf-11, Otf11, Skin, Skin-1a, Skn-1a, Skn-li Location Chromosome 9: 43,123,939-43,210,369 reverse strand. GRCm38:CM001002.2 About this gene This gene has 3 transcripts (splice variants), 199 orthologues, 16 paralogues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pou2f3-202 ENSMUST00000176636.4 2522 431aa ENSMUSP00000135115.1 Protein coding CCDS40596 H3BJT4 TSL:1 GENCODE basic APPRIS P2 Pou2f3-201 ENSMUST00000034513.12 2610 419aa ENSMUSP00000034513.5 Protein coding - Q3U5D1 TSL:1 GENCODE basic APPRIS ALT2 Pou2f3-203 ENSMUST00000213862.1 2181 No protein - Retained intron - - TSL:5 106.43 kb Forward strand 43.12Mb 43.14Mb 43.16Mb 43.18Mb 43.20Mb 43.22Mb Genes Rpl26-ps6-201 >processed pseudogene (Comprehensive set... Contigs < AC110169.7 AC173346.1 > Genes < Tmem136-201protein coding (Comprehensive set... < Tmem136-203lncRNA < Tmem136-202protein coding < Tmem136-204protein coding < Pou2f3-201protein coding < Pou2f3-202protein coding < Pou2f3-203retained intron Regulatory Build 43.12Mb 43.14Mb 43.16Mb 43.18Mb 43.20Mb 43.22Mb Reverse strand 106.43 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 processed transcript pseudogene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000176636 < Pou2f3-202protein coding Reverse strand 81.82 kb ENSMUSP00000135... MobiDB lite Low complexity (Seg) Superfamily Homeobox-like domain superfamily Lambda repressor-like, DNA-binding domain superfamily SMART POU-specific domain Homeobox domain Prints POU domain Octamer-binding transcription factor Pfam POU-specific domain Homeobox domain PROSITE profiles POU-specific domain Homeobox domain PROSITE patterns POU-specific domain Homeobox, conserved site POU-specific domain PANTHER PTHR11636 PTHR11636:SF81 Gene3D 1.10.260.40 1.10.10.60 CDD Homeobox domain All sequence SNPs/i..
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