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Mouse Wdr45b Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Wdr45b conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Wdr45b gene (NCBI Reference Sequence: NM_025793 ; Ensembl: ENSMUSG00000025173 ) is located on Mouse chromosome 11. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000026173). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Wdr45b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-266D20 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:
Exon 4 starts from about 23.74% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2469 bp, and the size of intron 4 for 3'-loxP site insertion: 1486 bp. The size of effective cKO region: ~588 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 10 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Wdr45b 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. 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 GC Content Distribution Window size: 300 bp
Sequence 12
Summary: Full Length(7088bp) | A(26.31% 1865) | C(20.01% 1418) | T(30.57% 2167) | G(23.11% 1638)
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% chr11 - 121338895 121341894 3000 browser details YourSeq 516 2357 2883 3000 99.1% chr7 - 90445571 90446104 534 browser details YourSeq 240 2341 2603 3000 95.8% chr13 + 92914183 92914464 282 browser details YourSeq 96 1 569 3000 75.5% chr1 - 156384191 156384550 360 browser details YourSeq 95 678 779 3000 97.1% chr16 - 11191027 11191129 103 browser details YourSeq 88 1 407 3000 91.6% chr17 - 8394835 8395275 441 browser details YourSeq 80 82 562 3000 85.9% chr12 + 86437380 86675001 237622 browser details YourSeq 62 247 407 3000 87.0% chrX + 157782760 157783148 389 browser details YourSeq 60 1 62 3000 98.4% chr1 - 178271061 178271122 62 browser details YourSeq 60 280 668 3000 70.0% chr2 + 172780496 172780647 152 browser details YourSeq 60 31 407 3000 72.1% chr19 + 5820013 5820237 225 browser details YourSeq 56 1 61 3000 96.8% chr4 - 59011168 59011235 68 browser details YourSeq 56 1 60 3000 96.7% chr4 - 33829029 33829088 60 browser details YourSeq 56 5 68 3000 96.7% chr5 + 148917301 148917366 66 browser details YourSeq 56 453 545 3000 80.5% chr1 + 175608571 175608664 94 browser details YourSeq 54 91 544 3000 64.5% chr18 + 35086667 35086926 260 browser details YourSeq 53 1990 2314 3000 89.6% chr1 - 120137408 120137899 492 browser details YourSeq 53 1 55 3000 98.2% chr3 + 142364388 142364442 55 browser details YourSeq 51 2181 2270 3000 76.7% chr2 + 102065789 102065866 78 browser details YourSeq 50 1 52 3000 98.1% chr5 - 134970021 134970072 52
Note: The 3000 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 3000 1 3000 3000 100.0% chr11 - 121335307 121338306 3000 browser details YourSeq 180 437 954 3000 97.4% chr12 - 110611155 110904738 293584 browser details YourSeq 176 2731 2924 3000 94.4% chr16 - 11190665 11190857 193 browser details YourSeq 173 415 680 3000 90.4% chr12 + 102444680 102444906 227 browser details YourSeq 172 346 595 3000 95.2% chr3 + 94617329 94617577 249 browser details YourSeq 167 322 595 3000 97.2% chr2 + 112400779 112401201 423 browser details YourSeq 165 415 595 3000 96.1% chr11 - 14117175 14117361 187 browser details YourSeq 164 415 617 3000 92.1% chr4 - 124622155 124622345 191 browser details YourSeq 163 331 595 3000 94.1% chr2 - 5843585 5844234 650 browser details YourSeq 163 415 595 3000 95.1% chr1 + 87694668 87694848 181 browser details YourSeq 161 415 595 3000 93.8% chr14 + 81656606 81656783 178 browser details YourSeq 160 415 595 3000 97.1% chr16 - 17031899 17032083 185 browser details YourSeq 160 415 595 3000 95.9% chr16 + 17919204 17919383 180 browser details YourSeq 160 415 595 3000 94.7% chr11 + 115735331 115735507 177 browser details YourSeq 159 349 595 3000 96.5% chr9 - 62333144 62333546 403 browser details YourSeq 159 408 595 3000 93.1% chr10 - 128080026 128080208 183 browser details YourSeq 158 415 595 3000 94.1% chr6 - 49065776 49065952 177 browser details YourSeq 158 415 595 3000 94.1% chr11 - 78078646 78078822 177 browser details YourSeq 158 415 595 3000 96.5% chr4_JH584294_random + 162314 162496 183 browser details YourSeq 158 415 595 3000 94.1% chr4 + 108846567 108846743 177
Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Wdr45b WD repeat domain 45B [ Mus musculus (house mouse) ] Gene ID: 66840, updated on 14-Aug-2019
Gene summary
Official Symbol Wdr45b provided by MGI Official Full Name WD repeat domain 45B provided by MGI Primary source MGI:MGI:1914090 See related Ensembl:ENSMUSG00000025173 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 WIPI-3; Wdr45l; AA408735; D16Bwg0193e; 0610008N23Rik Expression Ubiquitous expression in limb E14.5 (RPKM 23.7), ovary adult (RPKM 19.0) and 28 other tissues See more Orthologs human all
Genomic context
Location: 11 E2; 11 85.24 cM See Wdr45b in Genome Data Viewer
Exon count: 11
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (121327203..121354447, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (121188517..121215761, complement)
Chromosome 11 - NC_000077.6
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Transcript information: This gene has 5 transcripts
Gene: Wdr45b ENSMUSG00000025173
Description WD repeat domain 45B [Source:MGI Symbol;Acc:MGI:1914090] Gene Synonyms 0610008N23Rik, D16Bwg0193e, Wdr45l Location Chromosome 11: 121,327,224-121,354,445 reverse strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 198 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Wdr45b-201 ENSMUST00000026173.12 2321 344aa ENSMUSP00000026173.6 Protein coding CCDS25773 Q9CR39 TSL:1 GENCODE basic APPRIS P1
Wdr45b-202 ENSMUST00000106110.9 702 218aa ENSMUSP00000101716.3 Protein coding - F7AZP7 CDS 3' incomplete TSL:5
Wdr45b-204 ENSMUST00000136797.2 616 172aa ENSMUSP00000119591.1 Protein coding - F6RIG8 CDS 3' incomplete TSL:3
Wdr45b-203 ENSMUST00000127373.1 2455 No protein - Retained intron - - TSL:1
Wdr45b-205 ENSMUST00000137230.1 918 No protein - lncRNA - - TSL:3
47.22 kb Forward strand 121.32Mb 121.33Mb 121.34Mb 121.35Mb 121.36Mb Contigs AL663088.10 > Genes (Comprehensive set... < Wdr45b-201protein coding < Rab40b-201protein coding
< Wdr45b-205lncRNA < Wdr45b-203retained intron < Rab40b-204lncRNA
< Wdr45b-202protein coding < Rab40b-202lncRNA
< Wdr45b-204protein coding < Rab40b-203lncRNA
Regulatory Build
121.32Mb 121.33Mb 121.34Mb 121.35Mb 121.36Mb Reverse strand 47.22 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
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Transcript: ENSMUST00000026173
< Wdr45b-201protein coding
Reverse strand 27.22 kb
ENSMUSP00000026... Low complexity (Seg) Superfamily WD40-repeat-containing domain superfamily SMART WD40 repeat PANTHER PTHR11227:SF18
PTHR11227 Gene3D WD40/YVTN repeat-like-containing domain superfamily
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend
missense variant synonymous variant
Scale bar 0 40 80 120 160 200 240 280 344
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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