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Mouse Fbxw11 Knockout Project (CRISPR/Cas9)

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Mouse Fbxw11 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Fbxw11 Knockout Project (CRISPR/Cas9) Objective: To create a Fbxw11 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fbxw11 gene (NCBI Reference Sequence: NM_134015 ; Ensembl: ENSMUSG00000020271 ) 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 13 (Transcript: ENSMUST00000076383). Exon 3~4 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 3 starts from about 8.76% of the coding region. Exon 3~4 covers 17.11% of the coding region. The size of effective KO region: ~3810 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 14 Legends Exon of mouse Fbxw11 Knockout region Page 2 of 9 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 3 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 4 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(24.1% 482) | C(23.2% 464) | T(28.65% 573) | G(24.05% 481) Note: The 2000 bp section upstream of Exon 3 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(25.6% 512) | C(20.3% 406) | T(31.7% 634) | G(22.4% 448) Note: The 2000 bp section downstream 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. Page 4 of 9 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 + 32706189 32708188 2000 browser details YourSeq 160 4 253 2000 81.5% chr4 - 130555404 130555652 249 browser details YourSeq 159 1 253 2000 81.8% chr2 - 147144958 147145216 259 browser details YourSeq 156 2 245 2000 85.8% chr7 + 122634812 122635057 246 browser details YourSeq 153 2 234 2000 86.0% chr1 + 57420712 57421066 355 browser details YourSeq 148 1 222 2000 85.6% chr8 - 79590121 79590343 223 browser details YourSeq 148 1 235 2000 81.7% chr1 + 125755049 125755284 236 browser details YourSeq 146 1 226 2000 85.6% chr1 + 166979279 166979503 225 browser details YourSeq 145 2 253 2000 86.5% chr7 - 131503377 131503630 254 browser details YourSeq 145 2 240 2000 83.2% chr18 + 81297165 81297404 240 browser details YourSeq 143 2 241 2000 84.6% chr12 + 109382692 109382933 242 browser details YourSeq 142 1 245 2000 83.0% chr3 - 87587294 87587540 247 browser details YourSeq 141 1 227 2000 81.6% chr7 + 64434935 64435151 217 browser details YourSeq 140 2 226 2000 83.8% chr14 - 122994281 122994509 229 browser details YourSeq 136 6 241 2000 81.9% chr6 + 116323294 116323524 231 browser details YourSeq 135 5 201 2000 85.1% chr18 - 12238104 12238302 199 browser details YourSeq 135 1 243 2000 83.8% chr15 + 5519245 5519477 233 browser details YourSeq 134 3 189 2000 86.2% chr6 - 29235567 29235752 186 browser details YourSeq 134 2 240 2000 83.2% chr12 - 28351364 28351605 242 browser details YourSeq 130 1 201 2000 84.0% chr4 - 131701249 131701449 201 Note: The 2000 bp section upstream of Exon 3 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 + 32711999 32713998 2000 browser details YourSeq 72 665 814 2000 76.4% chr2 + 32857497 32857638 142 browser details YourSeq 69 641 816 2000 89.8% chr1 + 90453550 90453726 177 browser details YourSeq 66 714 820 2000 91.4% chr14 + 118329977 118330089 113 browser details YourSeq 65 705 807 2000 93.6% chr4 - 42824996 42825108 113 browser details YourSeq 64 714 818 2000 87.3% chr4 - 19593844 19593950 107 browser details YourSeq 61 705 807 2000 93.2% chr4_JH584293_random - 180984 181096 113 browser details YourSeq 61 705 807 2000 93.2% chr4_GL456350_random - 121273 121385 113 browser details YourSeq 61 705 807 2000 93.2% chr4 - 42499633 42499745 113 browser details YourSeq 61 652 796 2000 84.5% chr2 + 29038064 29038234 171 browser details YourSeq 60 682 817 2000 81.5% chr11 + 4789164 4789309 146 browser details YourSeq 58 714 816 2000 85.6% chr16 - 23371641 23371749 109 browser details YourSeq 58 640 819 2000 90.3% chr4 + 118059228 118059437 210 browser details YourSeq 57 751 820 2000 91.2% chr3 - 8652504 8652573 70 browser details YourSeq 57 628 817 2000 77.7% chr1 - 136794620 136794801 182 browser details YourSeq 54 1887 1973 2000 81.7% chr1 - 194691616 194691715 100 browser details YourSeq 51 747 807 2000 96.6% chr4_JH584294_random - 172799 172867 69 browser details YourSeq 51 733 797 2000 90.8% chr4 - 43333051 43333121 71 browser details YourSeq 51 751 817 2000 90.5% chr16 - 92192420 92192487 68 browser details YourSeq 50 726 798 2000 82.3% chr19 - 38752029 38752095 67 Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Fbxw11 F-box and WD-40 domain protein 11 [ Mus musculus (house mouse) ] Gene ID: 103583, updated on 27-Aug-2019 Gene summary Official Symbol Fbxw11 provided by MGI Official Full Name F-box and WD-40 domain protein 11 provided by MGI Primary source MGI:MGI:2144023 See related Ensembl:ENSMUSG00000020271 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 HOS; BTRC2; BTRCP2; Fbxw1b; AA536858; 2310065A07Rik Expression Ubiquitous expression in cerebellum adult (RPKM 24.5), whole brain E14.5 (RPKM 24.2) and 28 other tissues See more Orthologs human all Genomic context Location: 11; 11 A4 See Fbxw11 in Genome Data Viewer Exon count: 15 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (32642555..32746816) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (32542875..32646816) Chromosome 11 - NC_000077.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Fbxw11 ENSMUSG00000020271 Description F-box and WD-40 domain protein 11 [Source:MGI Symbol;Acc:MGI:2144023] Gene Synonyms 2310065A07Rik, BTRC2, BTRCP2, Fbxw1b, HOS Location Chromosome 11: 32,642,724-32,746,816 forward strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 225 orthologues, 27 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Fbxw11- ENSMUST00000076383.7 4019 563aa ENSMUSP00000075721.7 Protein coding CCDS36125 Q5SRY7 TSL:1 201 GENCODE basic APPRIS P3 Fbxw11- ENSMUST00000093205.12 3938 542aa ENSMUSP00000090893.6 Protein coding CCDS70157 Q5SRY7 TSL:1 202 GENCODE basic APPRIS ALT1 Fbxw11- ENSMUST00000109366.7 3821 529aa ENSMUSP00000104991.1 Protein coding CCDS70158 Q5SRY7 TSL:1 203 GENCODE basic APPRIS ALT1 Fbxw11- ENSMUST00000143290.1 620 43aa ENSMUSP00000116834.1 Nonsense mediated - F7BGU3 CDS 5' 205 decay incomplete TSL:3 Fbxw11- ENSMUST00000137898.1 309 No - lncRNA - - TSL:1 204 protein Page 7 of 9 https://www.alphaknockout.com 124.09 kb Forward strand 32.64Mb 32.66Mb 32.68Mb 32.70Mb 32.72Mb 32.74Mb Genes Fbxw11-203 >protein coding (Comprehensive set... Fbxw11-204 >lncRNA Fbxw11-202 >protein coding Fbxw11-201 >protein coding Fbxw11-205 >nonsense mediated decay Contigs AL669844.20 > AL669951.18 > Regulatory Build 32.64Mb 32.66Mb 32.68Mb 32.70Mb 32.72Mb 32.74Mb Reverse strand 124.09 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000076383 103.94 kb Forward strand Fbxw11-201 >protein coding ENSMUSP00000075... Superfamily F-box-like domain superfamily WD40-repeat-containing domain superfamily SMART F-box domain WD40 repeat D domain of beta-TrCP Prints G-protein beta WD-40 repeat Pfam D domain of beta-TrCP WD40 repeat F-box domain PROSITE profiles F-box domain WD40-repeat-containing domain WD40 repeat PROSITE patterns WD40 repeat, conserved site PANTHER PTHR44129 PTHR44129:SF4 Gene3D 1.20.1280.50 WD40/YVTN repeat-like-containing domain superfamily CDD cd00200 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 563 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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