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Mouse Me1 Knockout Project (CRISPR/Cas9)

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Mouse Me1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Me1 Knockout Project (CRISPR/Cas9) Objective: To create a Me1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Me1 gene (NCBI Reference Sequence: NM_001198933 ; Ensembl: ENSMUSG00000032418 ) is located on Mouse chromosome 9. 14 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 14 (Transcript: ENSMUST00000185374). Exon 2~3 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: Mice homozygous for a spontaneous allele exhibit decreased body weight on a medium fat diet, altered cytoplasmic malic enzyme activity, and a male-specific reduction in food intake on a high fat diet. Exon 2 starts from about 1.15% of the coding region. Exon 2~3 covers 17.15% of the coding region. The size of effective KO region: ~2089 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 14 Legends Exon of mouse Me1 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. 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 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. 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(23.2% 464) | C(21.2% 424) | T(32.7% 654) | G(22.9% 458) 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(23.1% 462) | C(22.35% 447) | T(34.7% 694) | G(19.85% 397) Note: The 2000 bp section downstream 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. 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% chr9 - 86679953 86681952 2000 browser details YourSeq 115 371 893 2000 74.5% chr4 + 52485400 52485809 410 browser details YourSeq 98 353 869 2000 77.8% chr14 + 57308907 57309312 406 browser details YourSeq 92 295 790 2000 86.0% chr2 + 128726000 128987575 261576 browser details YourSeq 85 300 694 2000 79.5% chr1 - 172036549 172036925 377 browser details YourSeq 81 292 790 2000 76.2% chr3 + 137735232 137735685 454 browser details YourSeq 80 374 871 2000 71.5% chr1 + 80495325 80495745 421 browser details YourSeq 78 262 376 2000 87.5% chr8 + 119244117 119244231 115 browser details YourSeq 66 300 395 2000 86.7% chr7 + 36251326 36251422 97 browser details YourSeq 65 366 592 2000 80.3% chr6 + 17311395 17311607 213 browser details YourSeq 65 274 771 2000 76.0% chr1 + 46970977 46971438 462 browser details YourSeq 64 248 398 2000 91.0% chrX - 139472125 139472639 515 browser details YourSeq 64 234 396 2000 89.2% chr10 + 90844439 90844676 238 browser details YourSeq 63 292 395 2000 89.9% chrX - 65726193 65726297 105 browser details YourSeq 62 366 518 2000 75.0% chr18 + 39143854 39144456 603 browser details YourSeq 61 302 398 2000 83.6% chr5 + 117557676 117557773 98 browser details YourSeq 60 304 398 2000 84.1% chr1 - 124525705 124525800 96 browser details YourSeq 60 330 483 2000 87.9% chr6 + 40152905 40153057 153 browser details YourSeq 59 515 668 2000 76.4% chr7 - 109502519 109502653 135 browser details YourSeq 58 459 824 2000 73.3% chr1 - 165189798 165190104 307 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% chr9 - 86675864 86677863 2000 browser details YourSeq 412 1030 1598 2000 89.6% chr19 - 7550370 7551001 632 browser details YourSeq 403 1024 1589 2000 89.1% chr13 - 17524937 17525572 636 browser details YourSeq 394 1030 1598 2000 90.5% chr2 - 181430421 181431037 617 browser details YourSeq 387 1025 1581 2000 88.2% chr19 - 47915699 47916293 595 browser details YourSeq 380 1015 1581 2000 88.6% chr10 + 3676758 3677364 607 browser details YourSeq 379 1030 1598 2000 91.5% chr11 - 74381111 74381729 619 browser details YourSeq 377 1030 1654 2000 90.2% chr9 - 37962981 37963646 666 browser details YourSeq 377 1047 1598 2000 90.3% chr6 + 34385337 34385940 604 browser details YourSeq 377 1027 1575 2000 90.3% chr10 + 129158219 129158792 574 browser details YourSeq 375 1043 1591 2000 89.1% chr12 + 49749415 49750039 625 browser details YourSeq 372 1025 1581 2000 89.3% chr6 + 13783654 13784238 585 browser details YourSeq 372 1030 1598 2000 90.2% chr16 + 49528376 49529009 634 browser details YourSeq 371 1016 1598 2000 86.3% chr2 + 143180637 143181291 655 browser details YourSeq 371 1016 1581 2000 88.3% chr1 + 30658784 30754549 95766 browser details YourSeq 369 1030 1581 2000 90.0% chr4 + 47394702 47395292 591 browser details YourSeq 368 1043 1581 2000 88.9% chr1 - 157236993 157237588 596 browser details YourSeq 366 1017 1581 2000 89.8% chr2 - 78699296 78699867 572 browser details YourSeq 366 1030 1596 2000 89.4% chr16 - 12858608 12859237 630 browser details YourSeq 364 1024 1581 2000 89.3% chr4 + 101823706 101824317 612 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Me1 malic enzyme 1, NADP(+)-dependent, cytosolic [ Mus musculus (house mouse) ] Gene ID: 17436, updated on 10-Oct-2019 Gene summary Official Symbol Me1 provided by MGI Official Full Name malic enzyme 1, NADP(+)-dependent, cytosolic provided by MGI Primary source MGI:MGI:97043 See related Ensembl:ENSMUSG00000032418 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 Mod1; Mdh-1; Mod-1; D9Ertd267e Expression Broad expression in subcutaneous fat pad adult (RPKM 140.3), mammary gland adult (RPKM 130.2) and 16 other tissues Orthologs See more human all Genomic context Location: 9 E3.1; 9 46.58 cM See Me1 in Genome Data Viewer Exon count: 16 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (86581363..86695914, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (86474970..86589947, complement) Chromosome 9 - NC_000075.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Me1 ENSMUSG00000032418 Description malic enzyme 1, NADP(+)-dependent, cytosolic [Source:MGI Symbol;Acc:MGI:97043] Gene Synonyms D9Ertd267e, Mdh-1, Mod-1, Mod1 Location Chromosome 9: 86,581,371-86,695,953 reverse strand. GRCm38:CM001002.2 About this gene This gene has 4 transcripts (splice variants), 208 orthologues, 2 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 Me1-202 ENSMUST00000185374.1 3292 552aa ENSMUSP00000140887.1 Protein coding CCDS81047 Q3TQP6 TSL:1 GENCODE basic Me1-201 ENSMUST00000034989.14 3288 572aa ENSMUSP00000034989.8 Protein coding CCDS23383 P06801 TSL:1 GENCODE basic APPRIS P1 Me1-204 ENSMUST00000189968.6 1055 No protein - lncRNA - - TSL:1 Me1-203 ENSMUST00000189847.1 254 No protein - lncRNA - - TSL:5 Page 7 of 9 https://www.alphaknockout.com 134.58 kb Forward strand 86.60Mb 86.65Mb 86.70Mb Genes Rwdd2a-203 >protein coding A330041J22Rik-201 >lncRNA (Comprehensive set... Rwdd2a-201 >protein coding Gm22138-201 >snoRNA Rwdd2a-202 >protein coding Contigs AC159811.2 > CT025628.2 > Genes (Comprehensive set... < Pgm3-201protein coding < Me1-203lncRNA < Gm38230-201TEC < Pgm3-202protein coding < Gm25949-201snRNA < Pgm3-203retained intron < Pgm3-204retained intron < Me1-201protein coding < Me1-202protein coding < Me1-204lncRNA Regulatory Build 86.60Mb 86.65Mb 86.70Mb Reverse strand 134.58 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000185374 < Me1-202protein coding Reverse strand 105.87 kb ENSMUSP00000140... Low complexity (Seg) Superfamily SSF53223 NAD(P)-binding domain superfamily SMART Malic enzyme, N-terminal domain Malic enzyme, NAD-binding Prints Malic oxidoreductase Pfam Malic enzyme, N-terminal domain Malic enzyme, NAD-binding PROSITE patterns Malic enzyme, conserved site PIRSF Malic oxidoreductase PANTHER PTHR23406 PTHR23406:SF17 Gene3D 1.20.1370.30 Malic enzyme, N-terminal domain superfamily 3.30.2330.30 3.40.50.720 CDD cd05312 All sequence SNPs/i..
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