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Mouse Ndufb3 Knockout Project (CRISPR/Cas9)

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Mouse Ndufb3 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ndufb3 Knockout Project (CRISPR/Cas9) Objective: To create a Ndufb3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ndufb3 gene (NCBI Reference Sequence: NM_025597 ; Ensembl: ENSMUSG00000026032 ) is located on Mouse chromosome 1. 3 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000027193). Exon 2~3 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 0.32% of the coding region. Exon 2~3 covers 100.0% of the coding region. The size of effective KO region: ~4711 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 Legends Exon of mouse Ndufb3 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of start codon 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 stop codon is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(29.75% 595) | C(17.65% 353) | T(36.0% 720) | G(16.6% 332) Note: The 2000 bp section upstream of start codon 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(28.45% 569) | C(20.5% 410) | T(30.35% 607) | G(20.7% 414) Note: The 2000 bp section downstream of stop codon 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr1 + 58589093 58591092 2000 browser details YourSeq 79 581 705 2000 87.0% chr17 + 27825886 27826012 127 browser details YourSeq 75 502 701 2000 81.7% chr1 - 20860470 20860835 366 browser details YourSeq 74 581 699 2000 81.1% chr12 - 54607252 54607355 104 browser details YourSeq 73 593 725 2000 80.9% chr9 + 98437459 98437584 126 browser details YourSeq 73 581 721 2000 84.2% chr10 + 82061745 82061887 143 browser details YourSeq 72 581 701 2000 85.3% chr8 + 6068981 6069100 120 browser details YourSeq 70 587 700 2000 85.8% chr13 - 48949926 48950041 116 browser details YourSeq 67 587 701 2000 86.7% chr14 - 28567850 28567965 116 browser details YourSeq 66 581 701 2000 83.0% chr14 + 49905280 49905402 123 browser details YourSeq 63 615 780 2000 82.7% chr15 + 12210630 12210957 328 browser details YourSeq 62 591 716 2000 78.9% chr11 - 70384275 70384393 119 browser details YourSeq 61 587 701 2000 82.7% chr1 + 171535819 171535937 119 browser details YourSeq 60 626 716 2000 83.4% chr16 - 17882170 17882261 92 browser details YourSeq 60 590 701 2000 78.9% chr13 - 55504055 55504170 116 browser details YourSeq 60 588 674 2000 88.4% chr11 + 106305366 106305459 94 browser details YourSeq 58 626 709 2000 91.5% chr11 - 77035352 77035436 85 browser details YourSeq 57 582 701 2000 89.1% chr11 + 17157770 17157897 128 browser details YourSeq 56 587 701 2000 82.3% chr15 + 80271474 80271592 119 browser details YourSeq 56 581 674 2000 92.2% chr11 + 18018592 18018686 95 Note: The 2000 bp section upstream of start codon 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% chr1 + 58595804 58597803 2000 browser details YourSeq 111 1 120 2000 96.7% chr17 - 12430825 12430945 121 browser details YourSeq 108 1 117 2000 96.6% chr12 - 85187398 85187515 118 browser details YourSeq 107 20 155 2000 89.8% chr5 - 104601536 104601672 137 browser details YourSeq 104 391 519 2000 88.0% chr18 - 64200157 64200282 126 browser details YourSeq 104 391 506 2000 94.9% chr12 - 85187469 85187584 116 browser details YourSeq 102 20 151 2000 90.0% chr5 - 104647147 104647283 137 browser details YourSeq 102 1 118 2000 92.3% chr11 - 6307391 6307507 117 browser details YourSeq 93 395 506 2000 89.1% chr14 + 18095971 18096080 110 browser details YourSeq 85 20 125 2000 90.6% chr5 + 104875259 104875365 107 browser details YourSeq 85 1713 1828 2000 92.4% chr1 + 181748072 181748201 130 browser details YourSeq 81 1602 1802 2000 79.0% chr8 - 107836306 107836502 197 browser details YourSeq 76 1624 1818 2000 75.0% chr1 - 59776722 59776896 175 browser details YourSeq 75 1675 1920 2000 69.3% chr8 - 86800237 86800398 162 browser details YourSeq 75 1713 1844 2000 88.7% chr18 - 50103322 50103457 136 browser details YourSeq 74 1613 1819 2000 81.4% chr11 - 68061150 68061357 208 browser details YourSeq 73 1 85 2000 93.0% chr18 - 64200132 64200216 85 browser details YourSeq 72 1616 1794 2000 76.8% chr10 - 58528674 58528834 161 browser details YourSeq 71 1652 1833 2000 88.6% chr12 + 77470309 77470672 364 browser details YourSeq 70 1713 1844 2000 77.5% chr18 + 47706974 47707096 123 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Ndufb3 NADH:ubiquinone oxidoreductase subunit B3 [ Mus musculus (house mouse) ] Gene ID: 66495, updated on 12-Aug-2019 Gene summary Official Symbol Ndufb3 provided by MGI Official Full Name NADH:ubiquinone oxidoreductase subunit B3 provided by MGI Primary source MGI:MGI:1913745 See related Ensembl:ENSMUSG00000026032 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 CI-B12; 2700033I16Rik Expression Ubiquitous expression in heart adult (RPKM 65.8), bladder adult (RPKM 47.1) and 26 other tissues See more Orthologs human all Genomic context Location: 1; 1 C1.3 See Ndufb3 in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (58586397..58595964) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (58643443..58652792) Chromosome 1 - NC_000067.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Ndufb3 ENSMUSG00000026032 Description NADH:ubiquinone oxidoreductase subunit B3 [Source:MGI Symbol;Acc:MGI:1913745] Gene Synonyms 2700033I16Rik Location Chromosome 1: 58,586,384-58,595,964 forward strand. GRCm38:CM000994.2 About this gene This gene has 1 transcript (splice variant), 206 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ndufb3-201 ENSMUST00000027193.8 763 104aa ENSMUSP00000027193.8 Protein coding CCDS14977 Q9CQZ6 TSL:1 GENCODE basic APPRIS P1 29.58 kb Forward strand 58.58Mb 58.59Mb 58.60Mb Genes (Comprehensive set... Ndufb3-201 >protein coding Contigs AC118698.7 > Genes < Fam126b-207protein coding (Comprehensive set... < Fam126b-202protein coding < Fam126b-201protein coding < Fam126b-206protein coding < Fam126b-205lncRNA < Fam126b-204retained intron < Fam126b-203retained intron Regulatory Build 58.58Mb 58.59Mb 58.60Mb Reverse strand 29.58 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 RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000027193 9.58 kb Forward strand Ndufb3-201 >protein coding ENSMUSP00000027... PDB-ENSP mappings Low complexity (Seg) Pfam NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3 PANTHER PTHR15082:SF2 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) YR M M R R Variant Legend inframe insertion missense variant synonymous variant Scale bar 0 10 20 30 40 50 60 70 80 90 104 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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