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Mouse Fbxw8 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Fbxw8 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fbxw8 gene (NCBI Reference Sequence: NM_172721 ; Ensembl: ENSMUSG00000032867 ) is located on Mouse chromosome 5. 11 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 11 (Transcript: ENSMUST00000049474). 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 Fbxw8 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-156D17 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 a null allele display partial late embryonic lethality with embryonic growth retardation and abnormal placental morphology.

Exon 4 starts from about 32.83% 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: 3784 bp, and the size of intron 4 for 3'-loxP site insertion: 11166 bp. The size of effective cKO region: ~589 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 11 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Fbxw8 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(7089bp) | A(21.91% 1553) | C(25.76% 1826) | T(27.39% 1942) | G(24.94% 1768)

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% chr5 - 118125275 118128274 3000 browser details YourSeq 59 2402 2490 3000 83.2% chr11 + 117547409 117547497 89 browser details YourSeq 58 2402 2480 3000 86.2% chr19 + 45827700 45827776 77 browser details YourSeq 58 2269 2480 3000 68.0% chr1 + 26212825 26212917 93 browser details YourSeq 57 2402 2496 3000 80.0% chr5 - 31065989 31066081 93 browser details YourSeq 57 2297 2480 3000 66.3% chr17 + 46670813 46670898 86 browser details YourSeq 55 2402 2480 3000 84.6% chr1 + 134261486 134261562 77 browser details YourSeq 54 2406 2480 3000 89.1% chr9 + 49063774 49063847 74 browser details YourSeq 54 2402 2480 3000 83.4% chr12 + 24967230 24967306 77 browser details YourSeq 53 2399 2480 3000 83.1% chr15 - 16344038 16344117 80 browser details YourSeq 53 2402 2778 3000 64.8% chr13 - 95653556 95653636 81 browser details YourSeq 52 2402 2480 3000 78.0% chr2 + 18232119 18232189 71 browser details YourSeq 51 2405 2480 3000 84.7% chr17 - 33657616 33657689 74 browser details YourSeq 51 2405 2480 3000 83.6% chr15 - 11297667 11297740 74 browser details YourSeq 51 2402 2480 3000 84.7% chr1 - 52103912 52103988 77 browser details YourSeq 51 2404 2480 3000 86.9% chr3 + 79217551 79217625 75 browser details YourSeq 51 2406 2486 3000 81.5% chr18 + 70633438 70633518 81 browser details YourSeq 51 2403 2478 3000 84.7% chr12 + 100819755 100819828 74 browser details YourSeq 50 2403 2479 3000 81.5% chr19 + 26912336 26912410 75 browser details YourSeq 50 2404 2478 3000 84.0% chr10 + 79974978 79975188 211

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% chr5 - 118121686 118124685 3000 browser details YourSeq 96 666 2212 3000 90.8% chr5 + 144386635 144405057 18423 browser details YourSeq 89 2120 2594 3000 75.0% chr12 + 79277904 79278189 286 browser details YourSeq 82 2115 2318 3000 74.0% chr10 - 111279964 111280080 117 browser details YourSeq 82 2114 2275 3000 81.2% chr11 + 28424491 28424639 149 browser details YourSeq 75 2115 2219 3000 88.7% chr10 - 62876307 62876414 108 browser details YourSeq 74 2114 2214 3000 85.5% chr19 - 29308341 29308439 99 browser details YourSeq 72 2114 2222 3000 78.8% chr18 - 10552844 10552942 99 browser details YourSeq 72 2115 2218 3000 89.3% chr12 - 23543041 23543163 123 browser details YourSeq 72 2115 2218 3000 89.3% chr12 - 22384021 22384143 123 browser details YourSeq 72 2113 2218 3000 84.9% chr17 + 46517191 46517295 105 browser details YourSeq 70 2122 2218 3000 88.1% chr11 - 46643791 46643896 106 browser details YourSeq 68 2114 2218 3000 89.5% chr13 + 33928580 33928689 110 browser details YourSeq 67 2114 2214 3000 81.9% chr1 - 176252197 176252293 97 browser details YourSeq 66 2131 2219 3000 84.9% chr15 - 91669891 91669977 87 browser details YourSeq 66 2131 2218 3000 94.7% chr1 + 94045861 94045953 93 browser details YourSeq 65 2142 2318 3000 72.0% chr1 + 185430149 185430243 95 browser details YourSeq 64 2117 2219 3000 80.7% chr11 - 23216825 23216922 98 browser details YourSeq 64 2117 2226 3000 84.1% chr11 + 3230637 3230746 110 browser details YourSeq 62 2116 2219 3000 76.0% chr16 - 31914251 31914336 86

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: Fbxw8 F-box and WD-40 domain protein 8 [ Mus musculus (house mouse) ] Gene ID: 231672, updated on 12-Aug-2019

Gene summary

Official Symbol Fbxw8 provided by MGI Official Full Name F-box and WD-40 domain protein 8 provided by MGI Primary source MGI:MGI:1923041 See related Ensembl:ENSMUSG00000032867 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 FBW6; FBW8; Fbx29; FBXO29; 4930438M06Rik Expression Ubiquitous expression in ovary adult (RPKM 39.6), subcutaneous fat pad adult (RPKM 36.4) and 28 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 F See Fbxw8 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (118064981..118155458, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (118514990..118605467, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 2 transcripts

Gene: Fbxw8 ENSMUSG00000032867

Description F-box and WD-40 domain protein 8 [Source:MGI Symbol;Acc:MGI:1923041] Gene Synonyms 4930438M06Rik, FBW6, FBW8, FBXO29, Fbx29 Location Chromosome 5: 118,064,965-118,155,464 reverse strand. GRCm38:CM000998.2 About this gene This gene has 2 transcripts (splice variants), 193 orthologues, 27 paralogues, is a member of 1 Ensembl protein family and is associated with 23 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fbxw8-201 ENSMUST00000049474.8 5000 598aa ENSMUSP00000047012.7 Protein coding CCDS19609 Q8BIA4 TSL:1 GENCODE basic APPRIS P1

Fbxw8-202 ENSMUST00000201545.1 1937 No protein - Retained intron - - TSL:1

110.50 kb Forward strand 118.06Mb 118.08Mb 118.10Mb 118.12Mb 118.14Mb 118.16Mb Genes Tesc-201 >protein coding Hrk-202 >lncRNA (Comprehensive set...

Tesc-202 >nonsense mediated decay

Tesc-203 >retained intron

Tesc-206 >lncRNGAm9754-201 >lncRNA

Contigs < AC114993.18 < AC110254.11 Genes (Comprehensive set... < Fbxw8-201protein coding

< Fbxw8-202retained intron

< Gm26411-201snRNA

Regulatory Build

118.06Mb 118.08Mb 118.10Mb 118.12Mb 118.14Mb 118.16Mb Reverse strand 110.50 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000049474

< Fbxw8-201protein coding

Reverse strand 90.50 kb

ENSMUSP00000047... MobiDB lite Low complexity (Seg) Superfamily F-box-like domain superfamily

WD40-repeat-containing domain superfamily SMART F-box domain WD40 repeat

Pfam F-box domain

PROSITE profiles F-box domain WD40 repeat

WD40-repeat-containing domain PROSITE patterns WD40 repeat, conserved site

PANTHER PTHR44019:SF11

PTHR44019 Gene3D 1.20.1280.50 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 60 120 180 240 300 360 420 480 598

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

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