https://www.alphaknockout.com Mouse Me1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Me1 conditional knockout Mouse model (C57BL/6N) 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 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Me1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-56G17 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: 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. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 7005 bp, and the size of intron 2 for 3'-loxP site insertion: 1805 bp. The size of effective cKO region: ~634 bp. The cKO 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 3 14 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Me1 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement 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 Summary: Full Length(7134bp) | A(24.56% 1752) | C(21.22% 1514) | T(33.14% 2364) | G(21.08% 1504) 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 8 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% chr9 - 86680203 86683202 3000 browser details YourSeq 115 1621 2143 3000 74.5% chr4 + 52485400 52485809 410 browser details YourSeq 98 1603 2119 3000 77.8% chr14 + 57308907 57309312 406 browser details YourSeq 85 1550 1944 3000 79.5% chr1 - 172036549 172036925 377 browser details YourSeq 81 1542 2040 3000 76.2% chr3 + 137735232 137735685 454 browser details YourSeq 80 1550 2126 3000 73.9% chr4 + 22455855 22456301 447 browser details YourSeq 80 1624 2121 3000 71.5% chr1 + 80495325 80495745 421 browser details YourSeq 78 1082 1727 3000 73.2% chr8 + 107797513 107797866 354 browser details YourSeq 70 1616 1829 3000 87.4% chr6 + 17311395 17311606 212 browser details YourSeq 66 1550 1645 3000 86.7% chr7 + 36251326 36251422 97 browser details YourSeq 66 1555 1885 3000 76.1% chr3 + 51891130 51891438 309 browser details YourSeq 65 1593 1732 3000 90.0% chr8 + 54824945 54825086 142 browser details YourSeq 65 1524 2021 3000 76.0% chr1 + 46970977 46971438 462 browser details YourSeq 64 1498 1648 3000 91.0% chrX - 139472125 139472639 515 browser details YourSeq 64 1530 1738 3000 94.6% chr7 + 117298119 117298375 257 browser details YourSeq 63 1542 1645 3000 89.9% chrX - 65726193 65726297 105 browser details YourSeq 62 1595 2040 3000 71.8% chr2 + 128987224 128987575 352 browser details YourSeq 62 1616 1768 3000 75.0% chr18 + 39143854 39144456 603 browser details YourSeq 60 1554 1648 3000 84.1% chr1 - 124525705 124525800 96 browser details YourSeq 60 1580 1733 3000 87.9% chr6 + 40152905 40153057 153 Note: The 3000 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 3000 1 3000 3000 100.0% chr9 - 86676569 86679568 3000 browser details YourSeq 211 2715 3000 3000 90.3% chr4 + 69626237 69626548 312 browser details YourSeq 209 2723 2998 3000 89.7% chr18 + 53489627 53489905 279 browser details YourSeq 208 2721 3000 3000 88.9% chr14 - 105594662 105594946 285 browser details YourSeq 207 2729 3000 3000 88.2% chr10 - 124186310 124186582 273 browser details YourSeq 204 2722 3000 3000 88.7% chr2 - 78699595 78699867 273 browser details YourSeq 201 2715 2999 3000 90.4% chr14 + 7691917 7692205 289 browser details YourSeq 201 2588 3000 3000 84.3% chr12 + 67126722 67127101 380 browser details YourSeq 201 2721 3000 3000 89.2% chr11 + 47390526 47390819 294 browser details YourSeq 200 2735 3000 3000 88.0% chr19 - 7550734 7551001 268 browser details YourSeq 199 2714 3000 3000 90.4% chr14 - 58127489 58127777 289 browser details YourSeq 199 2724 3000 3000 88.8% chr2 + 106130080 106130357 278 browser details YourSeq 199 2720 3000 3000 88.2% chr10 + 3676758 3677041 284 browser details YourSeq 198 2730 3000 3000 88.2% chr19 - 47916023 47916293 271 browser details YourSeq 198 2718 3000 3000 89.5% chr6 + 90554720 90555005 286 browser details YourSeq 197 2716 2983 3000 88.8% chrX - 72974485 72974762 278 browser details YourSeq 197 2741 3000 3000 91.3% chr16 + 37924685 37924944 260 browser details YourSeq 197 2752 3000 3000 90.6% chr11 + 9862196 9862445 250 browser details YourSeq 195 2714 2999 3000 85.7% chr18 - 10427599 10427922 324 browser details YourSeq 194 2722 2983 3000 89.2% chr6 - 23761492 23761753 262 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 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 5 of 8 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 6 of 8 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 7 of 8 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... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 552 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.