https://www.alphaknockout.com Mouse Cyb561 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Cyb561 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cyb561 gene (NCBI Reference Sequence: NM_007805 ; Ensembl: ENSMUSG00000019590 ) is located on Mouse chromosome 11. 6 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000019734). Exon 2~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cyb561 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-250E22 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: Exon 2~6 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 6. The size of intron 1 for 5'-loxP site insertion: 6270 bp. The size of effective cKO region: ~2723 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele T gRNA region G 5' A 3' 1 2 3 4 5 6 Targeting vector T G A Targeted allele T G A Constitutive KO allele (After Cre recombination) Legends Exon of mouse Cyb561 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 Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(8950bp) | A(21.51% 1925) | C(23.96% 2144) | T(26.06% 2332) | G(28.48% 2549) 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% chr11 - 105938078 105941077 3000 browser details YourSeq 24 538 563 3000 96.2% chr2 + 106323191 106323216 26 browser details YourSeq 24 507 530 3000 100.0% chr13 + 93177214 93177237 24 browser details YourSeq 24 2664 2689 3000 96.2% chr10 + 71043811 71043836 26 browser details YourSeq 22 2646 2667 3000 100.0% chr19 + 53214446 53214467 22 browser details YourSeq 21 41 61 3000 100.0% chr6 - 128128181 128128201 21 browser details YourSeq 21 85 105 3000 100.0% chr4 - 62690976 62690996 21 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% chr11 - 105932378 105935377 3000 browser details YourSeq 171 2294 2712 3000 82.4% chr17 - 26395395 26395699 305 browser details YourSeq 152 2294 2679 3000 88.0% chr5 - 31960410 31960978 569 browser details YourSeq 149 2295 2679 3000 89.5% chr5 + 143491758 143492340 583 browser details YourSeq 148 2294 2677 3000 92.7% chr14 + 76581554 76581953 400 browser details YourSeq 134 2555 2731 3000 91.1% chr4 - 106445534 106445731 198 browser details YourSeq 130 2335 2700 3000 81.3% chr9 + 78112532 78112794 263 browser details YourSeq 129 2555 2731 3000 89.2% chr19 - 24791116 24791305 190 browser details YourSeq 122 2338 2679 3000 90.7% chr10 - 91190520 91191091 572 browser details YourSeq 120 2583 2727 3000 92.5% chr16 - 33564140 33564289 150 browser details YourSeq 118 2555 2723 3000 91.1% chr7 + 116996361 116996543 183 browser details YourSeq 116 2555 2731 3000 91.6% chr16 + 14134234 14134428 195 browser details YourSeq 115 2555 2700 3000 92.1% chr7 - 142958808 142958966 159 browser details YourSeq 113 2583 2724 3000 91.4% chr7 - 129672801 129672951 151 browser details YourSeq 112 2555 2732 3000 90.6% chr16 - 33552380 33552573 194 browser details YourSeq 112 2222 2679 3000 81.0% chr6 + 113162707 113162896 190 browser details YourSeq 109 2555 2694 3000 93.1% chr10 + 42611164 42611319 156 browser details YourSeq 108 2589 2731 3000 91.7% chr4 + 136687371 136687513 143 browser details YourSeq 107 2588 2734 3000 89.4% chrX - 38215915 38216060 146 browser details YourSeq 107 2555 2683 3000 94.4% chr2 - 159883403 159883541 139 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Cyb561 cytochrome b-561 [ Mus musculus (house mouse) ] Gene ID: 13056, updated on 24-Oct-2019 Gene summary Official Symbol Cyb561 provided by MGI Official Full Name cytochrome b-561 provided by MGI Primary source MGI:MGI:103253 See related Ensembl:ENSMUSG00000019590 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 Expression Biased expression in genital fat pad adult (RPKM 144.4), adrenal adult (RPKM 27.6) and 12 other tissues See more Orthologs human all Genomic context Location: 11 E1; 11 68.81 cM See Cyb561 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (105933702..105945019, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (105795018..105805461, complement) Chromosome 11 - NC_000077.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Cyb561 ENSMUSG00000019590 Description cytochrome b-561 [Source:MGI Symbol;Acc:MGI:103253] Location Chromosome 11: 105,933,702-105,953,336 reverse strand. GRCm38:CM001004.2 About this gene This gene has 7 transcripts (splice variants), 193 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 63 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cyb561-201 ENSMUST00000019734.10 2741 250aa ENSMUSP00000019734.4 Protein coding CCDS25542 Q60720 TSL:1 GENCODE basic APPRIS P1 Cyb561-206 ENSMUST00000184086.7 1130 192aa ENSMUSP00000138931.1 Protein coding - V9GX11 TSL:5 GENCODE basic Cyb561-204 ENSMUST00000150563.2 967 99aa ENSMUSP00000138838.1 Protein coding - V9GWU6 CDS 3' incomplete TSL:3 Cyb561-205 ENSMUST00000183493.7 886 106aa ENSMUSP00000139125.1 Protein coding - V9GXF4 CDS 5' incomplete TSL:5 Cyb561-202 ENSMUST00000143251.7 816 209aa ENSMUSP00000121990.1 Protein coding - A2A685 CDS 3' incomplete TSL:3 Cyb561-207 ENSMUST00000184269.2 771 181aa ENSMUSP00000138889.1 Protein coding - V9GWY3 CDS 3' incomplete TSL:5 Cyb561-203 ENSMUST00000146607.1 505 No protein - lncRNA - - TSL:2 Page 6 of 8 https://www.alphaknockout.com 39.63 kb Forward strand 105.93Mb 105.94Mb 105.95Mb 105.96Mb Genes Tanc2-202 >protein coding Gm9910-201 >TEC (Comprehensive set... Tanc2-204 >protein coding Tanc2-201 >protein coding Contigs AL596246.10 > Genes (Comprehensive set... < Cyb561-201protein coding < Cyb561-205protein coding < Cyb561-206protein coding < Cyb561-203lncRNA < Cyb561-202protein coding < Cyb561-207protein coding < Cyb561-204protein coding Regulatory Build 105.93Mb 105.94Mb 105.95Mb 105.96Mb Reverse strand 39.63 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000019734 < Cyb561-201protein coding Reverse strand 10.71 kb ENSMUSP00000019... Transmembrane heli... Low complexity (Seg) SMART Cytochrome b561/ferric reductase transmembrane Pfam Cytochrome b561/ferric reductase transmembrane PROSITE profiles Cytochrome b561/ferric reductase transmembrane PANTHER Cytochrome b561 PTHR10106 Gene3D 1.20.120.1770 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 250 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.