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Mouse Dusp18 Knockout Project (CRISPR/Cas9)

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Mouse Dusp18 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Dusp18 Knockout Project (CRISPR/Cas9) Objective: To create a Dusp18 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Dusp18 gene (NCBI Reference Sequence: NM_173745 ; Ensembl: ENSMUSG00000047205 ) is located on Mouse chromosome 11. 2 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 2 (Transcript: ENSMUST00000055931). Exon 2 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 2 starts from about 0.18% of the coding region. Exon 2 covers 84.75% of the coding region. The size of effective KO region: ~478 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 gRNA region 5' gRNA region 3' 1 2 Legends Exon of mouse Dusp18 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 2 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 stop codon 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 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(23.65% 473) | C(26.5% 530) | T(27.1% 542) | G(22.75% 455) Note: The 2000 bp section upstream of Exon 2 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(25.65% 513) | C(24.6% 492) | T(26.6% 532) | G(23.15% 463) Note: The 2000 bp section downstream of stop codon 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% chr11 + 3895099 3897098 2000 browser details YourSeq 37 730 769 2000 97.5% chr12 + 87074191 87074321 131 browser details YourSeq 34 731 770 2000 84.3% chr11 + 92381749 92381786 38 browser details YourSeq 31 731 769 2000 87.9% chr1 - 117501736 117501772 37 browser details YourSeq 30 739 770 2000 90.4% chr15 + 30153096 30153126 31 browser details YourSeq 28 722 756 2000 76.7% chr6 + 134714593 134714622 30 browser details YourSeq 28 1185 1221 2000 89.2% chr18 + 42193427 42193464 38 browser details YourSeq 28 730 770 2000 68.8% chr14 + 35061749 35061781 33 browser details YourSeq 26 731 762 2000 82.8% chr3 - 26149112 26149141 30 browser details YourSeq 23 734 759 2000 83.4% chr14 - 31410048 31410071 24 Note: The 2000 bp section upstream of Exon 2 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 + 3897577 3899576 2000 browser details YourSeq 97 194 1619 2000 90.6% chr1 - 152692230 152737007 44778 browser details YourSeq 76 1575 1772 2000 88.6% chr9 - 60551930 60552128 199 browser details YourSeq 62 1585 1777 2000 91.9% chr2 + 107163569 107163956 388 browser details YourSeq 62 1580 1711 2000 86.5% chr1 + 153303233 153303362 130 browser details YourSeq 61 1602 1774 2000 82.0% chr14 - 76178543 76178707 165 browser details YourSeq 61 1595 1769 2000 80.7% chr19 + 43944294 43944473 180 browser details YourSeq 60 1581 1748 2000 89.8% chr6 + 124933058 124933223 166 browser details YourSeq 59 1506 1704 2000 75.0% chr9 - 75543865 75544036 172 browser details YourSeq 57 1601 1755 2000 92.6% chr1 - 72199978 72200142 165 browser details YourSeq 57 1691 1769 2000 87.4% chr1 + 59800411 59800896 486 browser details YourSeq 53 1690 1871 2000 93.5% chr1 + 87816793 87884096 67304 browser details YourSeq 51 1592 1742 2000 92.0% chr19 + 44481806 44481967 162 browser details YourSeq 50 1603 1754 2000 93.2% chr9 - 73191725 73191898 174 browser details YourSeq 50 1581 1770 2000 93.0% chr7 + 133918109 133918301 193 browser details YourSeq 48 1089 1197 2000 92.9% chr8 - 64172273 64172381 109 browser details YourSeq 48 1689 1764 2000 93.0% chr7 - 46898750 46898826 77 browser details YourSeq 48 1601 1770 2000 90.0% chr3 - 95178765 95178939 175 browser details YourSeq 47 1581 1702 2000 79.3% chr9 - 99202488 99202597 110 browser details YourSeq 47 1691 1764 2000 86.2% chr2 - 180921710 180921787 78 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Dusp18 dual specificity phosphatase 18 [ Mus musculus (house mouse) ] Gene ID: 75219, updated on 12-Aug-2019 Gene summary Official Symbol Dusp18 provided by MGI Official Full Name dual specificity phosphatase 18 provided by MGI Primary source MGI:MGI:1922469 See related Ensembl:ENSMUSG00000047205 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 DSP18; DUSP20; AI430870; BB028064; LMWDSP20; 4930527G07Rik Expression Broad expression in heart adult (RPKM 4.6), testis adult (RPKM 2.8) and 21 other tissues See more Orthologs human all Genomic context Location: 11; 11 A1 See Dusp18 in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (3894935..3901299) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (3795243..3801299) Chromosome 11 - NC_000077.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Dusp18 ENSMUSG00000047205 Description dual specificity phosphatase 18 [Source:MGI Symbol;Acc:MGI:1922469] Gene Synonyms 4930527G07Rik, DSP18, DUSP20, LMWDSP20 Location Chromosome 11: 3,895,240-3,901,296 forward strand. GRCm38:CM001004.2 About this gene This gene has 2 transcripts (splice variants), 73 orthologues, 31 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Dusp18-201 ENSMUST00000055931.4 4632 188aa ENSMUSP00000057346.4 Protein coding CCDS24369 Q8VE01 TSL:1 GENCODE basic APPRIS P1 Dusp18-202 ENSMUST00000109996.7 2685 188aa ENSMUSP00000105624.1 Protein coding CCDS24369 Q8VE01 TSL:5 GENCODE basic APPRIS P1 26.06 kb Forward strand 3.89Mb 3.90Mb 3.91Mb Genes (Comprehensive set... Dusp18-201 >protein coding Dusp18-202 >protein coding Contigs AL731853.9 > Genes < 4921536K21Rik-201protein coding < Slc35e4-202protein coding (Comprehensive set... < Slc35e4-201protein coding Regulatory Build 3.89Mb 3.90Mb 3.91Mb Reverse strand 26.06 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000055931 6.06 kb Forward strand Dusp18-201 >protein coding ENSMUSP00000057... Superfamily Protein-tyrosine phosphatase-like SMART Dual specificity protein phosphatase domain Prints Atypical dual specificity phosphatase Atypical dual specificity phosphatase, subfamily B Pfam Dual specificity phosphatase, catalytic domain PROSITE profiles Dual specificity protein phosphatase domain Tyrosine specific protein phosphatases domain PROSITE patterns Protein-tyrosine phosphatase, active site PANTHER PTHR46495 PTHR46495:SF2 Gene3D Protein-tyrosine phosphatase-like CDD cd14573 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 188 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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