https://www.alphaknockout.com Mouse Dio2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Dio2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Dio2 gene (NCBI Reference Sequence: NM_010050 ; Ensembl: ENSMUSG00000007682 ) is located on Mouse chromosome 12. 2 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 2 (Transcript: ENSMUST00000082432). 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 Dio2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-82B20 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 knock-out allele display elevated thyroxine (T4) and thyroid-stimulating hormone levels, changes in the metabolism and excretion of iodothyronines, and impaired adaptive thermogenesis. Exon 2 covers 72.18% of the coding region. Start codon is in exon 1, and stop codon is in exon 2. The size of intron 1 for 5'-loxP site insertion: 8072 bp. The size of effective cKO region: ~849 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele T A 5' gRNA region G 3' 1 2 Targeting vector T A G Targeted allele T A G Constitutive KO allele (After Cre recombination) Legends Exon of mouse Dio2 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(7076bp) | A(30.53% 2160) | C(19.7% 1394) | T(29.13% 2061) | G(20.65% 1461) 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% chr12 - 90730241 90733240 3000 browser details YourSeq 69 2485 2655 3000 88.1% chr1 - 23361477 23361668 192 browser details YourSeq 69 2473 2580 3000 91.7% chr10 + 86711403 86711523 121 browser details YourSeq 66 2493 2607 3000 79.1% chr11 + 105086495 105086594 100 browser details YourSeq 65 2484 2581 3000 92.4% chr5 + 130403757 130403857 101 browser details YourSeq 64 2492 2578 3000 95.8% chr15 + 77979092 77979178 87 browser details YourSeq 62 2493 2580 3000 85.9% chr12 + 17673221 17673306 86 browser details YourSeq 60 2491 2582 3000 80.6% chr8 - 117485470 117485547 78 browser details YourSeq 60 2492 2579 3000 86.9% chr4 - 141337352 141337438 87 browser details YourSeq 60 2492 2581 3000 89.5% chr10 - 62138834 62359137 220304 browser details YourSeq 60 2496 2581 3000 91.0% chr5 + 136543360 136543443 84 browser details YourSeq 59 2484 2579 3000 89.5% chr13 - 38622409 38622505 97 browser details YourSeq 58 2492 2579 3000 87.5% chr5 - 147585150 147585236 87 browser details YourSeq 58 2490 2581 3000 82.5% chr4 - 125271711 125271800 90 browser details YourSeq 57 2497 2700 3000 76.2% chr6 - 114236240 114236386 147 browser details YourSeq 56 2493 2581 3000 95.3% chr7 - 129940720 129940808 89 browser details YourSeq 56 2494 2581 3000 88.3% chr4 + 148409824 148409910 87 browser details YourSeq 55 2491 2698 3000 72.4% chr8 - 125079878 125080009 132 browser details YourSeq 55 2502 2581 3000 91.1% chr17 - 45083766 45083847 82 browser details YourSeq 55 2495 2588 3000 83.2% chr18 + 21295431 21295523 93 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% chr12 - 90726415 90729414 3000 browser details YourSeq 32 2615 2649 3000 87.9% chr10 - 95344411 95344443 33 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Dio2 deiodinase, iodothyronine, type II [ Mus musculus (house mouse) ] Gene ID: 13371, updated on 10-Oct-2019 Gene summary Official Symbol Dio2 provided by MGI Official Full Name deiodinase, iodothyronine, type II provided by MGI Primary source MGI:MGI:1338833 See related Ensembl:ENSMUSG00000007682 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as 5DII; DIOII; AI324267 Summary The protein encoded by this gene belongs to the iodothyronine deiodinase family. It catalyzes the conversion of Expression prohormone thyroxine (3,5,3',5'-tetraiodothyronine, T4) to the bioactive thyroid hormone (3,5,3'-triiodothyronine, T3) by outer ring 5'-deiodination. This gene is highly expressed in brain, placenta and mammary gland. It is thought to be responsible for the 'local' production of T3, and thus important in influencing thyroid hormone action in these tissues. Knockout studies in mice suggest that this gene may play an important role in brown adipose tissue lipogenesis, auditory function, and bone formation. This protein is a selenoprotein containing the non-standard amino acid, selenocysteine (Sec), which is encoded by the UGA codon that normally signals translation termination. The 3' UTRs of selenoprotein mRNAs contain a conserved stem-loop structure, designated the Sec insertion sequence (SECIS) element, that is necessary for the recognition of UGA as a Sec codon, rather than as a stop signal. Unlike the other two members (DIO1 and DIO3) of this enzyme family, the mRNA for this gene contains an additional in-frame UGA codon that has been reported (in human) to function either as a Sec or a stop codon, resulting in two potential isoforms with one or two Sec residues; however, only the upstream Sec (conserved with the single Sec residue found at the active site in DIO1 and DIO3) was shown to be essential for enzyme activity (PMID:10403186). In addition, the lack of conservation of the protein extension past the second TGA codon suggests that the one-Sec containing isoform represents the canonical form. [provided by RefSeq, Oct 2018] Orthologs Biased expression in subcutaneous fat pad adult (RPKM 14.7), frontal lobe adult (RPKM 8.9) and 4 other tissuesS ee more human all Genomic context Location: 12; 12 D3 See Dio2 in Genome Data Viewer Exon count: 2 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (90724552..90739038, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (91962992..91976878, complement) Chromosome 12 - NC_000078.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Dio2 ENSMUSG00000007682 Description deiodinase, iodothyronine, type II [Source:MGI Symbol;Acc:MGI:1338833] Location Chromosome 12: 90,724,552-90,738,438 reverse strand. GRCm38:CM001005.2 About this gene This gene has 1 transcript (splice variant), 205 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Dio2-201 ENSMUST00000082432.4 5815 266aa ENSMUSP00000081013.3 Protein coding CCDS36514 Q9Z1Y9 TSL:1 GENCODE basic APPRIS P1 33.89 kb Forward strand 90.72Mb 90.73Mb 90.74Mb Genes Gm26512-201 >lncRNA (Comprehensive set... Gm26512-203 >lncRNA Gm26512-202 >lncRNA Contigs < CT573100.6 Genes (Comprehensive set... < Dio2-201protein coding Regulatory Build 90.72Mb 90.73Mb 90.74Mb Reverse strand 33.89 kb Regulation Legend CTCF Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000082432 < Dio2-201protein coding Reverse strand 13.89 kb ENSMUSP00000081... Superfamily Thioredoxin-like superfamily Pfam Iodothyronine deiodinase PROSITE patterns Iodothyronine deiodinase, active site PIRSF Iodothyronine deiodinase PANTHER PTHR11781:SF20 Iodothyronine deiodinase Gene3D 3.40.30.10 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 266 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.