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Mouse Septin10 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Septin10 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Septin10 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Septin10 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Septin10 gene (NCBI Reference Sequence: NM_001024911.2 ; Ensembl: ENSMUSG00000019917 ) is located on Mouse chromosome 10. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000165971). 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 Septin10 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-368J21 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 26.0% 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: 8454 bp, and the size of intron 4 for 3'-loxP site insertion: 2343 bp. The size of effective cKO region: ~687 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 10 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Septin10 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com 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(7187bp) | A(28.54% 2051) | C(18.45% 1326) | T(33.25% 2390) | G(19.76% 1420) 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. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr10 - 59183962 59186961 3000 browser details YourSeq 730 765 1577 3000 95.1% chr2 - 171913186 171913995 810 browser details YourSeq 728 765 1577 3000 95.7% chr14 + 20822657 20823470 814 browser details YourSeq 723 768 1577 3000 95.1% chrX + 151216938 151217747 810 browser details YourSeq 723 765 1577 3000 94.7% chr17 + 11429902 11430707 806 browser details YourSeq 721 765 1577 3000 95.4% chr17 - 33429250 33430064 815 browser details YourSeq 721 767 1577 3000 95.1% chr14 - 76028984 76029797 814 browser details YourSeq 718 763 1577 3000 94.9% chr8 - 24325034 24325851 818 browser details YourSeq 715 768 1577 3000 94.7% chr15 + 8800231 8801043 813 browser details YourSeq 715 764 1577 3000 95.1% chr10 + 84452552 84453366 815 browser details YourSeq 714 766 1577 3000 95.4% chr8 - 35247409 35248220 812 browser details YourSeq 714 763 1577 3000 94.6% chr14 - 66889859 66890669 811 browser details YourSeq 714 765 1577 3000 94.5% chr11 - 48665076 48665889 814 browser details YourSeq 712 765 1577 3000 95.1% chr12 + 73418229 73419039 811 browser details YourSeq 711 749 1577 3000 93.3% chr6 + 107158059 107158867 809 browser details YourSeq 711 744 1577 3000 93.9% chr13 + 94538602 94539413 812 browser details YourSeq 710 765 1570 3000 95.5% chr19 - 15281166 15281971 806 browser details YourSeq 710 730 1577 3000 92.5% chr15 - 54823461 54824288 828 browser details YourSeq 710 766 1577 3000 94.8% chr10 - 126047280 126048090 811 browser details YourSeq 710 768 1577 3000 94.3% chr7 + 76064783 76065595 813 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% chr10 - 59180275 59183274 3000 browser details YourSeq 21 1185 1205 3000 100.0% chr16 - 98024707 98024727 21 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Septin10 septin 10 [ Mus musculus (house mouse) ] Gene ID: 103080, updated on 26-Jun-2020 Gene summary Official Symbol Septin10 provided by MGI Official Full Name septin 10 provided by MGI Primary source MGI:MGI:1918110 See related Ensembl:ENSMUSG00000019917 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 Sept10; AA408298; AI874685; 4921515A04Rik; 9430099J10Rik Expression Broad expression in testis adult (RPKM 14.3), bladder adult (RPKM 8.0) and 23 other tissues See more Orthologs human all Genomic context Location: 10; 10 B4 See Septin10 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (59141624..59221500, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (58604375..58684595, complement) Chromosome 10 - NC_000076.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Septin10 ENSMUSG00000019917 Description septin 10 [Source:MGI Symbol;Acc:MGI:1918110] Gene Synonyms 4921515A04Rik, 9430099J10Rik, Sept10 Location Chromosome 10: 59,141,627-59,221,847 reverse strand. GRCm38:CM001003.2 About this gene This gene has 3 transcripts (splice variants), 303 orthologues, 15 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 Septin10-201 ENSMUST00000165971.2 2532 427aa ENSMUSP00000129023.1 Protein coding CCDS48566 A0A0R4J233 TSL:1 GENCODE basic APPRIS P2 Septin10-203 ENSMUST00000220156.1 1821 452aa ENSMUSP00000151293.1 Protein coding - A0A1W2P6J7 TSL:1 GENCODE basic APPRIS ALT2 Septin10-202 ENSMUST00000218171.1 1051 196aa ENSMUSP00000151948.1 Protein coding - A0A1W2P866 CDS 5' incomplete TSL:3 100.22 kb Forward strand 59.14Mb 59.16Mb 59.18Mb 59.20Mb 59.22Mb Genes Sh3rf3-205 >protein coding Gm23223-201 >snoRNA Sowahc-201 >protein coding (Comprehensive set... Contigs AC159909.8 > AC153941.3 > Genes (Comprehensive set... < Septin10-203protein coding < Septin10-201protein coding < Septin10-202protein coding < AC153941.1-201processed pseudogene Regulatory Build 59.14Mb 59.16Mb 59.18Mb 59.20Mb 59.22Mb Reverse strand 100.22 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000165971 < Septin10-201protein coding Reverse strand 57.73 kb ENSMUSP00000129... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily P-loop containing nucleoside triphosphate hydrolase Pfam Septin-type guanine nucleotide-binding (G) domain PROSITE profiles Septin-type guanine nucleotide-binding (G) domain PIRSF Septin PANTHER PTHR18884 PTHR18884:SF50 Gene3D 3.40.50.300 CDD Septin All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 427 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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