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Mouse Degs2 Knockout Project (CRISPR/Cas9)

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Mouse Degs2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Degs2 Knockout Project (CRISPR/Cas9) Objective: To create a Degs2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Degs2 gene (NCBI Reference Sequence: NM_001171002 ; Ensembl: ENSMUSG00000021263 ) is located on Mouse chromosome 12. 3 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 3 (Transcript: ENSMUST00000167978). 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 9.67% of the coding region. Exon 2 covers 86.6% of the coding region. The size of effective KO region: ~743 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 2 3 Legends Exon of mouse Degs2 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 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. 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(21.5% 430) | C(26.9% 538) | T(28.2% 564) | G(23.4% 468) 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(21.4% 428) | C(22.7% 454) | T(25.35% 507) | G(30.55% 611) Note: The 2000 bp section downstream 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. 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% chr12 - 108692637 108694636 2000 browser details YourSeq 143 416 800 2000 95.6% chr1 + 51489507 51571443 81937 browser details YourSeq 123 382 524 2000 93.8% chr10 - 36535071 36535218 148 browser details YourSeq 116 384 526 2000 92.1% chr12 - 62516924 62517070 147 browser details YourSeq 111 382 523 2000 93.8% chr12 - 100903464 100903614 151 browser details YourSeq 111 383 524 2000 90.5% chr10 + 12109917 12110061 145 browser details YourSeq 110 383 511 2000 95.2% chr12 - 60157082 60157221 140 browser details YourSeq 110 384 520 2000 91.7% chr18 + 34390803 34390943 141 browser details YourSeq 109 380 503 2000 95.8% chr17 - 71607059 71607186 128 browser details YourSeq 109 383 524 2000 92.3% chr4 + 106340527 106340672 146 browser details YourSeq 109 387 524 2000 91.0% chr12 + 87772950 87773091 142 browser details YourSeq 108 383 524 2000 89.8% chr1 - 174779620 174779766 147 browser details YourSeq 108 382 526 2000 91.6% chr16 + 4004918 4005066 149 browser details YourSeq 106 394 524 2000 90.8% chr11 - 78930190 78930322 133 browser details YourSeq 106 383 520 2000 90.3% chr2 + 60096051 60096193 143 browser details YourSeq 106 383 525 2000 92.8% chr10 + 82269086 82269232 147 browser details YourSeq 104 380 805 2000 78.7% chr13 + 77315494 77315626 133 browser details YourSeq 104 380 527 2000 87.3% chr11 + 94793482 94793631 150 browser details YourSeq 104 383 511 2000 92.0% chr11 + 70383387 70383519 133 browser details YourSeq 103 380 524 2000 92.6% chr13 + 55525092 55525426 335 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% chr12 - 108689894 108691893 2000 browser details YourSeq 69 636 711 2000 97.3% chr15 + 74266434 74266621 188 browser details YourSeq 65 606 695 2000 82.5% chr3 + 154398124 154398198 75 browser details YourSeq 65 636 704 2000 97.2% chr3 + 119069814 119069882 69 browser details YourSeq 61 636 723 2000 95.9% chr7 - 34590981 34591110 130 browser details YourSeq 59 507 690 2000 96.9% chr1 - 42947334 42947518 185 browser details YourSeq 58 636 703 2000 92.7% chrX + 69875237 69875304 68 browser details YourSeq 56 578 704 2000 93.8% chr1 + 15486189 15750704 264516 browser details YourSeq 52 657 722 2000 95.0% chr7 + 111229950 111230053 104 browser details YourSeq 51 647 722 2000 91.7% chr3 + 131778154 131778246 93 browser details YourSeq 51 636 703 2000 93.3% chr14 + 27217282 27217349 68 browser details YourSeq 48 663 722 2000 94.5% chr3 - 94738929 94738991 63 browser details YourSeq 46 636 705 2000 84.5% chr19 - 40306502 40306569 68 browser details YourSeq 46 636 690 2000 85.5% chrX + 64758060 64758108 49 browser details YourSeq 45 650 706 2000 91.1% chr1 + 41257491 41257551 61 browser details YourSeq 43 655 700 2000 97.8% chr5 - 35890858 35890907 50 browser details YourSeq 43 666 711 2000 91.0% chr4 - 33605670 33605713 44 browser details YourSeq 42 656 709 2000 88.9% chr12 - 25621698 25621751 54 browser details YourSeq 37 636 673 2000 100.0% chr8 - 126524507 126524545 39 browser details YourSeq 37 672 722 2000 75.0% chr7 - 98168382 98168425 44 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Degs2 delta(4)-desaturase, sphingolipid 2 [ Mus musculus (house mouse) ] Gene ID: 70059, updated on 12-Aug-2019 Gene summary Official Symbol Degs2 provided by MGI Official Full Name delta(4)-desaturase, sphingolipid 2 provided by MGI Primary source MGI:MGI:1917309 See related Ensembl:ENSMUSG00000021263 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 DES2; AI852933; 2210008A03Rik Expression Broad expression in large intestine adult (RPKM 53.1), duodenum adult (RPKM 52.2) and 20 other tissues See more Orthologs human all Genomic context Location: 12; 12 F1 See Degs2 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (108686792..108702306, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (109925002..109940516, complement) Chromosome 12 - NC_000078.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Degs2 ENSMUSG00000021263 Description delta(4)-desaturase, sphingolipid 2 [Source:MGI Symbol;Acc:MGI:1917309] Gene Synonyms 2210008A03Rik, Des2 Location Chromosome 12: 108,678,711-108,702,315 reverse strand. GRCm38:CM001005.2 About this gene This gene has 3 transcripts (splice variants), 191 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Degs2-202 ENSMUST00000167978.8 1729 286aa ENSMUSP00000125891.1 Protein coding CCDS49170 Q8R2F2 TSL:1 GENCODE basic Degs2-201 ENSMUST00000021691.5 1287 323aa ENSMUSP00000021691.4 Protein coding CCDS26162 Q8R2F2 TSL:1 GENCODE basic APPRIS P1 Degs2-203 ENSMUST00000222255.1 737 98aa ENSMUSP00000152122.1 Protein coding - A0A1Y7VIS8 CDS 5' incomplete TSL:3 Page 7 of 9 https://www.alphaknockout.com 43.60 kb Forward strand 108.67Mb 108.68Mb 108.69Mb 108.70Mb 108.71Mb Genes Evl-202 >protein coding Gm33467-201 >lncRNA (Comprehensive set... Evl-201 >protein coding Evl-203 >protein coding Evl-206 >protein coding Evl-204 >protein coding Evl-205 >retained intron Evl-207 >protein coding Contigs < AC139568.5 Genes (Comprehensive set... < Degs2-203protein coding < Degs2-202protein coding < Degs2-201protein coding Regulatory Build 108.67Mb 108.68Mb 108.69Mb 108.70Mb 108.71Mb Reverse strand 43.60 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000167978 < Degs2-202protein coding Reverse strand 14.06 kb ENSMUSP00000125... Low complexity (Seg) SMART Sphingolipid delta4-desaturase, N-terminal Pfam Sphingolipid delta4-desaturase, N-terminal Fatty acid desaturase domain PIRSF Sphingolipid delta4-desaturase PANTHER PTHR12879:SF14 PTHR12879 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 240 286 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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