https://www.alphaknockout.com

Mouse Cyb5r3 Knockout Project (CRISPR/Cas9)

Objective: To create a Cyb5r3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cyb5r3 (NCBI Reference Sequence: NM_029787 ; Ensembl: ENSMUSG00000018042 ) is located on Mouse 15. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000018186). Exon 2~8 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note:

Exon 2 starts from about 2.44% of the coding region. Exon 2~8 covers 78.85% of the coding region. The size of effective KO region: ~7381 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 5 6 7 8 9

Legends Exon of mouse Cyb5r3 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 2 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 8 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(21.2% 424) | C(22.7% 454) | T(23.7% 474) | G(32.4% 648)

Note: The 2000 bp section upstream of Exon 2 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(22.8% 456) | C(25.35% 507) | T(22.55% 451) | G(29.3% 586)

Note: The 2000 bp section downstream of Exon 8 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr15 - 83166207 83168206 2000 browser details YourSeq 39 1714 1775 2000 95.4% chr12 + 26486998 26487067 70 browser details YourSeq 33 1729 1764 2000 97.3% chr7 + 35379140 35379182 43 browser details YourSeq 32 1729 1763 2000 97.2% chr4 + 130213113 130213161 49 browser details YourSeq 29 1713 1756 2000 96.8% chr7 + 6801494 6801538 45 browser details YourSeq 29 1734 1763 2000 100.0% chr5 + 135834685 135834721 37 browser details YourSeq 29 1740 1781 2000 74.2% chr11 + 84806824 84806855 32 browser details YourSeq 28 1741 1777 2000 96.7% chr8 + 104505940 104505983 44 browser details YourSeq 28 1739 1768 2000 96.7% chr2 + 59029867 59029896 30 browser details YourSeq 27 1689 1750 2000 64.6% chr6 + 38864205 38864244 40 browser details YourSeq 27 1715 1757 2000 89.7% chr17 + 36253851 36253892 42 browser details YourSeq 27 1735 1763 2000 96.6% chr1 + 125177869 125177897 29 browser details YourSeq 26 1687 1723 2000 96.5% chr18 - 10545694 10545732 39 browser details YourSeq 26 1737 1763 2000 100.0% chr2 + 172413219 172413246 28 browser details YourSeq 25 1740 1764 2000 100.0% chrX - 36938837 36938861 25 browser details YourSeq 25 1739 1763 2000 100.0% chr8 - 56571045 56571069 25 browser details YourSeq 25 1740 1764 2000 100.0% chr5 - 139933426 139933450 25 browser details YourSeq 25 1740 1764 2000 100.0% chr5 - 136387453 136387477 25 browser details YourSeq 25 1739 1763 2000 100.0% chr2 - 156657063 156657087 25 browser details YourSeq 25 1740 1764 2000 100.0% chr2 - 117505080 117505104 25

Note: The 2000 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 2000 1 2000 2000 100.0% chr15 - 83156826 83158825 2000 browser details YourSeq 60 632 710 2000 94.2% chr5 - 100795100 100795182 83 browser details YourSeq 55 623 710 2000 82.4% chr12 - 20347809 20347892 84 browser details YourSeq 55 623 710 2000 82.4% chr12 + 21856000 21856083 84 browser details YourSeq 50 624 710 2000 89.1% chr11 - 63843225 63843313 89 browser details YourSeq 50 628 722 2000 83.4% chr10 - 77253272 77253361 90 browser details YourSeq 49 629 729 2000 77.1% chr12 - 24320615 24320702 88 browser details YourSeq 49 629 729 2000 77.1% chr12 - 23593145 23593232 88 browser details YourSeq 49 629 729 2000 77.1% chr12 - 23383915 23384002 88 browser details YourSeq 49 628 710 2000 83.1% chr10 - 63143361 63143439 79 browser details YourSeq 48 667 728 2000 88.5% chr6 - 47822290 47822347 58 browser details YourSeq 47 655 728 2000 75.4% chr15 - 85990116 85990184 69 browser details YourSeq 47 628 706 2000 85.5% chr13 - 100915588 100915663 76 browser details YourSeq 43 669 729 2000 84.4% chr13 - 38123259 38123316 58 browser details YourSeq 43 629 729 2000 75.5% chr12 - 21865565 21865652 88 browser details YourSeq 41 673 728 2000 95.7% chr16 - 16898129 16898201 73 browser details YourSeq 41 667 728 2000 90.4% chr10 + 60553959 60554022 64 browser details YourSeq 40 633 710 2000 79.7% chr16 - 10880915 10880989 75 browser details YourSeq 40 655 710 2000 86.4% chr14 - 25910345 25910397 53 browser details YourSeq 40 655 710 2000 86.4% chr14 - 26050095 26050147 53

Note: The 2000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cyb5r3 3 [ Mus musculus (house mouse) ] Gene ID: 109754, updated on 21-Oct-2019

Gene summary

Official Symbol Cyb5r3 provided by MGI Official Full Name cytochrome b5 reductase 3 provided by MGI Primary source MGI:MGI:94893 See related Ensembl:ENSMUSG00000018042 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 B5R; Dia1; Dia-1; C85115; WU:Cyb5r3; 0610016L08Rik; 2500002N19Rik Expression Broad expression in duodenum adult (RPKM 409.4), adrenal adult (RPKM 270.7) and 23 other tissues See more Orthologs human all

Genomic context

Location: 15 E1; 15 39.4 cM See Cyb5r3 in Genome Data Viewer Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (83153495..83176440, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (82983931..83002638, complement)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 3 transcripts

Gene: Cyb5r3 ENSMUSG00000018042

Description cytochrome b5 reductase 3 [Source:MGI Symbol;Acc:MGI:94893] Gene Synonyms 0610016L08Rik, 2500002N19Rik, Dia-1, Dia1 Location Chromosome 15: 83,153,494-83,172,592 reverse strand. GRCm38:CM001008.2 About this gene This gene has 3 transcripts (splice variants), 198 orthologues, 5 paralogues, 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

Cyb5r3- ENSMUST00000018186.15 2422 301aa ENSMUSP00000018186.9 Protein CCDS27698 Q9DCN2 TSL:1 201 coding GENCODE basic APPRIS P1

Cyb5r3- ENSMUST00000162178.7 2547 313aa ENSMUSP00000125636.1 Protein - F2Z456 TSL:1 202 coding GENCODE basic

Cyb5r3- ENSMUST00000162834.2 1982 278aa ENSMUSP00000124062.2 Protein - A0A0A0MQM3 TSL:5 203 coding Q9DCN2 GENCODE basic

39.10 kb Forward strand 83.15Mb 83.16Mb 83.17Mb 83.18Mb Rnu12-201 >snRNA Gm49463-201 >lncRNA (Comprehensive set...

Contigs < AL591952.9 Genes (Comprehensive set... < Poldip3-201protein coding < Cyb5r3-201protein coding < 7530414M10Rik-201lncRNA

< Poldip3-202protein coding < Cyb5r3-203protein coding

< Poldip3-203protein coding < Cyb5r3-202protein coding

< Poldip3-205retained intron

< Poldip3-206retained intron

Regulatory Build

83.15Mb 83.16Mb 83.17Mb 83.18Mb Reverse strand 39.10 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000018186

< Cyb5r3-201protein coding

Reverse strand 19.10 kb

ENSMUSP00000018... Low complexity (Seg) Superfamily Ferredoxin-NADP reductase (FNR), nucleotide-binding domain

Riboflavin synthase-like beta-barrel Prints NADH:cytochrome b5 reductase-like

Flavoprotein pyridine nucleotide cytochrome reductase Pfam pyridine nucleotide cytochrome reductase-like, FAD-binding domain

Oxidoreductase FAD/NAD(P)-binding PROSITE profiles FAD-binding domain, ferredoxin reductase-type PANTHER PTHR19370

PTHR19370:SF121 Gene3D 2.40.30.10 Ferredoxin-NADP reductase (FNR), nucleotide-binding domain

CDD cd06183

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 301

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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