https://www.alphaknockout.com Mouse Scg3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Scg3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Scg3 gene (NCBI Reference Sequence: NM_009130 ; Ensembl: ENSMUSG00000032181 ) is located on Mouse chromosome 9. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000034699). 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 Scg3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-337J17 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: Homozygous knockout causes dysregulation of the secretion of active peptide hormones from endocrine cells, exacerbating the adverse effects of inadequate diet (obesity, diabetes) and stress conditions. Exon 2 starts from about 6.51% 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: 644 bp, and the size of intron 2 for 3'-loxP site insertion: 618 bp. The size of effective cKO region: ~553 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 4 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Scg3 cKO region loxP site Page 2 of 8 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(7053bp) | A(29.38% 2072) | C(20.64% 1456) | T(30.75% 2169) | G(19.23% 1356) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. 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 - 75683241 75686240 3000 browser details YourSeq 52 28 96 3000 84.7% chr5 - 96860035 96860101 67 browser details YourSeq 51 736 858 3000 87.2% chr14 + 67113347 67113489 143 browser details YourSeq 49 29 99 3000 84.6% chr10 - 108485370 108485440 71 browser details YourSeq 47 730 790 3000 92.5% chr8 - 36757990 36758049 60 browser details YourSeq 46 26 99 3000 81.1% chr1 - 176019069 176019142 74 browser details YourSeq 45 726 796 3000 79.0% chr4 + 106599524 106599587 64 browser details YourSeq 44 25 96 3000 80.6% chr12 - 106349251 106349322 72 browser details YourSeq 43 9 70 3000 93.9% chr1 - 111604999 111605081 83 browser details YourSeq 43 9 70 3000 89.1% chr15 + 62195158 62195220 63 browser details YourSeq 41 26 74 3000 91.9% chr9 - 111767101 111767149 49 browser details YourSeq 41 9 66 3000 86.3% chr14 - 46494442 46494498 57 browser details YourSeq 36 698 800 3000 97.5% chr9 - 74044662 74044771 110 browser details YourSeq 36 26 69 3000 91.0% chr2 + 104578184 104578227 44 browser details YourSeq 36 26 202 3000 95.0% chr17 + 11760851 11761027 177 browser details YourSeq 35 26 70 3000 88.9% chr13 - 73375853 73375897 45 browser details YourSeq 35 26 70 3000 88.9% chr11 - 53885208 53885252 45 browser details YourSeq 35 28 70 3000 90.7% chr15 + 9238114 9238156 43 browser details YourSeq 34 56 99 3000 88.7% chr14 - 70866733 70866776 44 browser details YourSeq 34 56 99 3000 83.8% chr10 - 53418056 53418098 43 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 - 75679688 75682687 3000 browser details YourSeq 43 1488 1532 3000 97.8% chr11 - 38822443 38822487 45 browser details YourSeq 38 1467 1729 3000 59.6% chr19 + 38789021 38789123 103 browser details YourSeq 31 40 76 3000 91.9% chr10 - 20093315 20093351 37 browser details YourSeq 31 1462 1509 3000 94.3% chr2 + 21566569 21566618 50 browser details YourSeq 30 97 130 3000 94.2% chr14 - 26512118 26512151 34 browser details YourSeq 30 1143 1190 3000 82.4% chr1 - 81917141 81917185 45 browser details YourSeq 29 1474 1514 3000 93.8% chr5 - 147195181 147195221 41 browser details YourSeq 29 2026 2056 3000 96.8% chr4 - 83603496 83603526 31 browser details YourSeq 29 97 131 3000 91.5% chr3 - 104144513 104144547 35 browser details YourSeq 29 962 992 3000 96.8% chr11 + 96793556 96793586 31 browser details YourSeq 27 1483 1509 3000 100.0% chrX - 66007151 66007177 27 browser details YourSeq 27 1460 1496 3000 79.5% chr3 - 109343832 109343866 35 browser details YourSeq 27 1467 1509 3000 96.6% chr14 + 60187824 60187867 44 browser details YourSeq 27 96 132 3000 86.5% chr11 + 85785925 85785961 37 browser details YourSeq 26 90 127 3000 80.6% chr11 + 73149641 73149677 37 browser details YourSeq 25 113 151 3000 82.1% chr19 - 55724556 55724594 39 browser details YourSeq 25 120 148 3000 96.3% chr17 - 62804474 62804503 30 browser details YourSeq 24 488 519 3000 96.2% chr18 - 82286861 82286892 32 browser details YourSeq 22 1938 1963 3000 92.4% chr4 - 11057836 11057861 26 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: Scg3 secretogranin III [ Mus musculus (house mouse) ] Gene ID: 20255, updated on 24-Oct-2019 Gene summary Official Symbol Scg3 provided by MGI Official Full Name secretogranin III provided by MGI Primary source MGI:MGI:103032 See related Ensembl:ENSMUSG00000032181 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 Chgd; SgIII; 1B1075; AI385542 Expression Biased expression in cerebellum adult (RPKM 14.7), cortex adult (RPKM 13.8) and 7 other tissuesS ee more Orthologs human all Genomic context Location: 9 D; 9 42.32 cM See Scg3 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (75643366..75685959, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (75491173..75531863, complement) Chromosome 9 - NC_000075.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Scg3 ENSMUSG00000032181 Description secretogranin III [Source:MGI Symbol;Acc:MGI:103032] Gene Synonyms 1B1075, Chgd, SgIII Location Chromosome 9: 75,643,189-75,684,056 reverse strand. GRCm38:CM001002.2 About this gene This gene has 7 transcripts (splice variants), 192 orthologues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Scg3- ENSMUST00000034699.7 2384 471aa ENSMUSP00000034699.6 Protein coding CCDS23347 P47867 TSL:1 201 GENCODE basic APPRIS P1 Scg3- ENSMUST00000213324.1 2152 466aa ENSMUSP00000149561.1 Protein coding - P47867 TSL:1 202 GENCODE basic Scg3- ENSMUST00000214244.1 537 84aa ENSMUSP00000151074.1 Protein coding - A0A1L1SV66 CDS 3' 205 incomplete TSL:2 Scg3- ENSMUST00000215603.1 813 51aa ENSMUSP00000152480.1 Nonsense mediated - A0A1Y7VLI5 CDS 5' 206 decay incomplete TSL:5 Scg3- ENSMUST00000213637.1 574 No - lncRNA - - TSL:3 203 protein Scg3- ENSMUST00000214176.1 563 No - lncRNA - - TSL:2 204 protein Scg3- ENSMUST00000215722.1 452 No - lncRNA - - TSL:3 207 protein Page 6 of 8 https://www.alphaknockout.com 60.87 kb Forward strand 75.64Mb 75.65Mb 75.66Mb 75.67Mb 75.68Mb 75.69Mb Genes Lysmd2-201 >protein coding Gm37871-201 >TEC (Comprehensive set... Lysmd2-203 >nonsense mediated decay Lysmd2-202 >lncRNA Contigs AC123832.4 > AC144940.5 > Genes (Comprehensive set... < Scg3-201protein coding < Scg3-202protein coding < Scg3-206nonsense mediated decay < Scg3-203lncRNA < Scg3-205protein coding < Scg3-204lncRNA < Scg3-207lncRNA < Gm3671-201processed pseudogene Regulatory Build 75.64Mb 75.65Mb 75.66Mb 75.67Mb 75.68Mb 75.69Mb Reverse strand 60.87 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000034699 < Scg3-201protein coding Reverse strand 40.87 kb ENSMUSP00000034... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Cleavage site (Sign... Pfam Secretogranin III PANTHER Secretogranin III All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 471 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.