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Mouse Pou2f3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Pou2f3 conditional 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 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pou2f3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-233D3 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO 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 5 starts from about 18.41% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1488 bp, and the size of intron 5 for 3'-loxP site insertion: 2421 bp. The size of effective cKO region: ~603 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 4 5 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pou2f3 Homology arm cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7103bp) | A(23.05% 1637) | C(24.55% 1744) | T(28.5% 2024) | G(23.91% 1698)

Note: The sequence of homologous arms and cKO region 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr9 - 43145572 43148571 3000 browser details YourSeq 379 1115 2940 3000 93.1% chr11 - 78331671 78809466 477796 browser details YourSeq 336 1220 2982 3000 95.0% chr17 - 35183371 35676413 493043 browser details YourSeq 316 1224 2916 3000 93.2% chr2 + 150368059 150632518 264460 browser details YourSeq 230 1054 1382 3000 95.3% chr18 + 40242561 40242937 377 browser details YourSeq 222 807 1373 3000 94.8% chr11 - 84163622 84164220 599 browser details YourSeq 212 1140 1381 3000 96.1% chr7 - 89071865 89072218 354 browser details YourSeq 175 1177 1377 3000 93.7% chrX - 111593938 111594134 197 browser details YourSeq 165 898 1381 3000 93.7% chr13 - 64217429 64217984 556 browser details YourSeq 164 1124 1390 3000 87.1% chr1 + 133062678 133062889 212 browser details YourSeq 162 1050 1373 3000 87.3% chr2 + 156548736 156548918 183 browser details YourSeq 160 1216 1397 3000 95.0% chr11 - 97304287 97304491 205 browser details YourSeq 160 1090 1381 3000 84.3% chr5 + 107349298 107349506 209 browser details YourSeq 160 961 1373 3000 87.1% chr2 + 119695040 119695293 254 browser details YourSeq 158 1203 1389 3000 94.0% chr7 + 127067874 127068093 220 browser details YourSeq 151 1125 1382 3000 87.7% chr7 + 102338425 102338586 162 browser details YourSeq 151 1216 1382 3000 95.8% chr7 + 80899305 80899477 173 browser details YourSeq 151 1216 1393 3000 95.3% chr10 + 34219867 34220050 184 browser details YourSeq 149 1219 1382 3000 95.8% chr13 - 67609870 67610050 181 browser details YourSeq 149 1209 1373 3000 95.8% chr4 + 41139291 41139778 488

Note: The 3000 bp section upstream of Exon 5 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 3000 1 3000 3000 100.0% chr9 - 43141969 43144968 3000 browser details YourSeq 82 2170 2255 3000 97.7% chr1 - 106888756 106888841 86 browser details YourSeq 47 1868 1918 3000 96.1% chr1 - 83229672 83229722 51 browser details YourSeq 45 1868 1918 3000 96.0% chr5 - 133706176 133706238 63 browser details YourSeq 27 2 28 3000 100.0% chr11 - 54012736 54012762 27 browser details YourSeq 26 1 27 3000 100.0% chr1 + 182915291 182915397 107 browser details YourSeq 25 4 28 3000 100.0% chrX + 150993015 150993039 25 browser details YourSeq 24 4 27 3000 100.0% chr2 + 168064695 168064718 24 browser details YourSeq 24 4 27 3000 100.0% chr18 + 9849725 9849748 24 browser details YourSeq 23 6 28 3000 100.0% chr9 + 21087371 21087393 23 browser details YourSeq 23 6 28 3000 100.0% chr4 + 46088374 46088396 23 browser details YourSeq 22 2115 2138 3000 95.9% chr3 + 87339577 87339600 24 browser details YourSeq 21 964 985 3000 100.0% chr8 + 51629840 51629862 23 browser details YourSeq 21 1915 1935 3000 100.0% chr3 + 61909566 61909586 21 browser details YourSeq 21 2 22 3000 100.0% chr13 + 100357496 100357516 21 browser details YourSeq 21 2614 2634 3000 100.0% chr1 + 84728076 84728096 21

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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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

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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

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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... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 431

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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