https://www.alphaknockout.com Mouse Sesn3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Sesn3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Sesn3 gene (NCBI Reference Sequence: NM_030261 ; Ensembl: ENSMUSG00000032009 ) is located on Mouse chromosome 9. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000208222). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Sesn3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-250B9 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: When fed a high fat diet, mice homozygous for a gene trap allele exhibit impaired glucose tolerance, insulin resistance, reduced hepatic glucose production, impaired adipocyte glucose uptake, increased hepatic steatosis, and decreased mitochondria in the liver. Exon 5 starts from about 35.64% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 4065 bp, and the size of intron 6 for 3'-loxP site insertion: 4054 bp. The size of effective cKO region: ~2245 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 5' gRNA region gRNA region 3' 1 5 6 10 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Sesn3 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(8745bp) | A(28.87% 2525) | C(19.81% 1732) | T(29.15% 2549) | G(22.17% 1939) 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% chr9 + 14311247 14314246 3000 browser details YourSeq 176 610 2116 3000 87.1% chr10 + 48438144 48479589 41446 browser details YourSeq 133 699 2116 3000 93.5% chr1 - 60948472 61348692 400221 browser details YourSeq 120 247 748 3000 77.5% chr13 - 96696134 96696296 163 browser details YourSeq 117 1998 2136 3000 89.2% chr1 + 192751950 192752080 131 browser details YourSeq 116 586 753 3000 87.8% chr1 - 60015450 60015617 168 browser details YourSeq 115 1998 2136 3000 89.9% chr1 - 152056399 152056530 132 browser details YourSeq 114 1998 2135 3000 92.0% chr18 + 21614639 21614796 158 browser details YourSeq 113 1998 2136 3000 93.9% chrX - 118775592 118775732 141 browser details YourSeq 112 244 752 3000 74.2% chr1 - 110857934 110858127 194 browser details YourSeq 111 247 752 3000 75.9% chr4 + 120818924 120819083 160 browser details YourSeq 110 1998 2137 3000 90.6% chr1 - 123538786 123538923 138 browser details YourSeq 109 2003 2118 3000 97.4% chrX - 41877080 41877197 118 browser details YourSeq 109 1998 2119 3000 95.1% chr2 + 23809420 23809543 124 browser details YourSeq 109 1998 2117 3000 95.8% chr11 + 12277194 12277315 122 browser details YourSeq 109 1998 2130 3000 91.7% chr10 + 49965983 49966127 145 browser details YourSeq 106 1998 2121 3000 95.0% chr1 - 83452687 83452810 124 browser details YourSeq 105 1998 2115 3000 96.5% chr9 + 66401762 66401880 119 browser details YourSeq 103 610 752 3000 87.2% chr1 - 36488959 36489103 145 browser details YourSeq 102 1998 2116 3000 91.5% chr1 + 46530305 46530422 118 Note: The 3000 bp section upstream of Exon 5 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 + 14316492 14319491 3000 browser details YourSeq 126 903 1609 3000 83.8% chr10 - 122980054 122996129 16076 browser details YourSeq 105 938 1598 3000 89.6% chr11 - 101454166 101638847 184682 browser details YourSeq 89 1475 1604 3000 87.7% chr1 - 180879595 180879724 130 browser details YourSeq 87 1475 1600 3000 85.6% chr11 + 120802298 120802422 125 browser details YourSeq 85 1481 1609 3000 92.2% chr1 - 21338671 21338799 129 browser details YourSeq 83 1475 1612 3000 88.5% chr1 + 168744800 168744936 137 browser details YourSeq 82 1473 1609 3000 86.7% chr2 - 94507944 94508079 136 browser details YourSeq 79 1481 1609 3000 82.6% chr10 - 92394165 92394287 123 browser details YourSeq 78 774 979 3000 80.2% chr12 + 110354385 110354578 194 browser details YourSeq 76 870 979 3000 86.6% chr11 + 64444885 64444996 112 browser details YourSeq 75 1475 1608 3000 86.0% chr7 - 5676960 5677092 133 browser details YourSeq 70 1475 1598 3000 85.8% chr18 - 16581320 16581443 124 browser details YourSeq 70 1475 1564 3000 89.2% chr11 - 117448760 117448847 88 browser details YourSeq 67 1475 1604 3000 86.6% chr13 - 35596934 35597061 128 browser details YourSeq 67 1475 1588 3000 83.4% chr16 + 90214589 90214701 113 browser details YourSeq 66 794 987 3000 92.3% chr6 - 144989269 144989704 436 browser details YourSeq 65 1473 1609 3000 76.2% chr2 - 39127846 39127979 134 browser details YourSeq 65 1517 1612 3000 89.4% chr11 - 117961331 117961424 94 browser details YourSeq 65 1513 1609 3000 90.2% chr1 - 141575211 141575664 454 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Sesn3 sestrin 3 [ Mus musculus (house mouse) ] Gene ID: 75747, updated on 12-Aug-2019 Gene summary Official Symbol Sesn3 provided by MGI Official Full Name sestrin 3 provided by MGI Primary source MGI:MGI:1922997 See related Ensembl:ENSMUSG00000032009 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as SEST3; BC003348; 5630400E15Rik Expression Ubiquitous expression in ovary adult (RPKM 13.1), thymus adult (RPKM 11.0) and 28 other tissues See more Orthologs human all Genomic context Location: 9; 9 A1 See Sesn3 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (14276301..14326134) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (14080745..14130578) Chromosome 9 - NC_000075.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Sesn3 ENSMUSG00000032009 Description sestrin 3 [Source:MGI Symbol;Acc:MGI:1922997] Gene Synonyms 5630400E15Rik, SEST3 Location Chromosome 9: 14,275,067-14,333,101 forward strand. GRCm38:CM001002.2 About this gene This gene has 7 transcripts (splice variants), 203 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Sesn3-204 ENSMUST00000208222.1 9125 492aa ENSMUSP00000146362.1 Protein coding CCDS22820 A2RSF4 Q9CYP7 TSL:1 GENCODE basic APPRIS P1 Sesn3-201 ENSMUST00000034507.8 1827 414aa ENSMUSP00000034507.8 Protein coding - A0A0J9YMZ5 TSL:5 GENCODE basic Sesn3-207 ENSMUST00000209187.1 1700 353aa ENSMUSP00000146784.1 Protein coding - A0A140LIE1 TSL:5 GENCODE basic Sesn3-206 ENSMUST00000209145.1 4178 No protein - Retained intron - - TSL:NA Sesn3-203 ENSMUST00000208142.1 1915 No protein - Retained intron - - TSL:NA Sesn3-202 ENSMUST00000207207.1 714 No protein - Retained intron - - TSL:5 Sesn3-205 ENSMUST00000209106.1 505 No protein - lncRNA - - TSL:5 78.03 kb Forward strand 14.28Mb 14.30Mb 14.32Mb 14.34Mb Genes (Comprehensive set... Sesn3-207 >protein coding Gm47326-201 >lncRNA Sesn3-201 >protein coding Sesn3-204 >protein coding Sesn3-205 >lncRNA Sesn3-203 >retained intron Sesn3-202 >retained intron Sesn3-206 >retained intron Contigs < AC154264.2 CT485606.7 > Regulatory Build 14.28Mb 14.30Mb 14.32Mb 14.34Mb Reverse strand 78.03 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 processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000208222 56.80 kb Forward strand Sesn3-204 >protein coding ENSMUSP00000146... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily AhpD-like Pfam Sestrin PANTHER Sestrin PTHR12474:SF4 Gene3D AhpD-like All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 492 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.