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Mouse Ndufb6 Knockout Project (CRISPR/Cas9)

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Mouse Ndufb6 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ndufb6 Knockout Project (CRISPR/Cas9) Objective: To create a Ndufb6 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ndufb6 gene (NCBI Reference Sequence: NM_001033305 ; Ensembl: ENSMUSG00000071014 ) is located on Mouse chromosome 4. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000095128). Exon 1~3 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 1 starts from about 0.26% of the coding region. Exon 1~3 covers 82.81% of the coding region. The size of effective KO region: ~6526 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 4 Legends Exon of mouse Ndufb6 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 Exon 3 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. 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(28.65% 573) | C(21.3% 426) | T(28.9% 578) | G(21.15% 423) 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(28.0% 560) | C(18.55% 371) | T(33.65% 673) | G(19.8% 396) Note: The 2000 bp section downstream of Exon 3 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% chr4 - 40279338 40281337 2000 browser details YourSeq 228 1 327 2000 84.2% chr9 + 53088553 53088864 312 browser details YourSeq 162 265 567 2000 84.7% chr7 + 27621956 27622209 254 browser details YourSeq 153 384 646 2000 85.7% chrX + 142382416 142382623 208 browser details YourSeq 153 384 586 2000 87.0% chr2 + 170202929 170203127 199 browser details YourSeq 150 384 568 2000 91.4% chr5 - 92175594 92175776 183 browser details YourSeq 150 331 568 2000 90.7% chr12 - 69369654 69370208 555 browser details YourSeq 149 384 568 2000 91.8% chr16 - 11265926 11266113 188 browser details YourSeq 148 12 555 2000 84.8% chr19 - 7465133 7465533 401 browser details YourSeq 147 386 568 2000 91.2% chr9 + 118367160 118367345 186 browser details YourSeq 147 384 568 2000 90.6% chr8 + 123286696 123286881 186 browser details YourSeq 147 379 585 2000 88.5% chr8 + 10981389 10981593 205 browser details YourSeq 145 381 567 2000 90.5% chrX - 140425083 140425271 189 browser details YourSeq 145 384 568 2000 91.1% chr4 + 133658536 133658726 191 browser details YourSeq 145 384 586 2000 87.5% chr17 + 31120794 31120990 197 browser details YourSeq 145 384 568 2000 89.9% chr15 + 99482876 99483059 184 browser details YourSeq 145 386 568 2000 89.1% chr14 + 105954395 105954574 180 browser details YourSeq 145 384 565 2000 88.9% chr11 + 86446580 86446755 176 browser details YourSeq 144 384 566 2000 87.8% chr5 - 140537375 140537547 173 browser details YourSeq 144 384 567 2000 88.6% chr2 - 117445291 117445471 181 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% chr4 - 40270810 40272809 2000 browser details YourSeq 201 928 1136 2000 99.6% chr9 + 108037651 108037864 214 browser details YourSeq 200 895 1122 2000 94.8% chr8 - 57734341 57734557 217 browser details YourSeq 197 929 1136 2000 99.5% chr18 + 34575681 34575892 212 browser details YourSeq 195 918 1124 2000 97.6% chr13 - 100525606 100525832 227 browser details YourSeq 195 927 1136 2000 96.7% chr12 - 84492588 84492803 216 browser details YourSeq 194 930 1136 2000 98.5% chr11 + 98876174 98876381 208 browser details YourSeq 192 932 1140 2000 98.5% chr2 - 75690375 75690591 217 browser details YourSeq 192 932 1124 2000 100.0% chr9 + 59376160 59376354 195 browser details YourSeq 192 932 1131 2000 98.5% chr5 + 100283909 100284108 200 browser details YourSeq 191 932 1127 2000 99.0% chr11 - 102389647 102389844 198 browser details YourSeq 191 930 1123 2000 99.5% chr4 + 45863866 45864060 195 browser details YourSeq 191 932 1127 2000 99.0% chr18 + 12745273 12745470 198 browser details YourSeq 190 932 1123 2000 99.5% chr8 - 71501382 71501573 192 browser details YourSeq 190 930 1122 2000 99.5% chr2 - 172968395 172968602 208 browser details YourSeq 190 923 1121 2000 98.0% chr14 - 44836771 44837018 248 browser details YourSeq 190 928 1121 2000 99.5% chr10 - 78478675 78478876 202 browser details YourSeq 190 930 1122 2000 99.5% chrX + 152557797 152558006 210 browser details YourSeq 190 930 1122 2000 99.5% chr5 + 31745266 31745463 198 browser details YourSeq 190 930 1122 2000 99.5% chr10 + 120936070 120936271 202 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Ndufb6 NADH:ubiquinone oxidoreductase subunit B6 [ Mus musculus (house mouse) ] Gene ID: 230075, updated on 12-Aug-2019 Gene summary Official Symbol Ndufb6 provided by MGI Official Full Name NADH:ubiquinone oxidoreductase subunit B6 provided by MGI Primary source MGI:MGI:2684983 See related Ensembl:ENSMUSG00000071014 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 Gm137; CI-B17 Summary This gene encodes a subunit of complex I (NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain. This Expression complex functions in electron transport and generation of a proton gradient across the inner mitochondrial membrane to drive ATP synthesis. Data from human cell lines suggests that the encoded subunit may be required for electron transfer activity. [provided by RefSeq, Aug 2015] Orthologs Ubiquitous expression in heart adult (RPKM 89.9), kidney adult (RPKM 75.1) and 28 other tissues See more human all Genomic context Location: 4; 4 A5 See Ndufb6 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (40270591..40279421, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (40217696..40226401, complement) Chromosome 4 - NC_000070.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Ndufb6 ENSMUSG00000071014 Description NADH:ubiquinone oxidoreductase subunit B6 [Source:MGI Symbol;Acc:MGI:2684983] Gene Synonyms LOC230075 Location Chromosome 4: 40,270,591-40,279,421 reverse strand. GRCm38:CM000997.2 About this gene This gene has 2 transcripts (splice variants), 194 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ndufb6-201 ENSMUST00000095128.9 659 128aa ENSMUSP00000092746.3 Protein coding CCDS18045 A2AP31 Q3UIU2 TSL:1 GENCODE basic APPRIS P1 Ndufb6-202 ENSMUST00000108108.2 480 97aa ENSMUSP00000103743.2 Protein coding - A2AP32 TSL:3 GENCODE basic 28.83 kb Forward strand 40.265Mb 40.270Mb 40.275Mb 40.280Mb 40.285Mb Genes Smim27-201 >protein coding (Comprehensive set... Smim27-202 >lncRNA Contigs AL831793.4 > Genes (Comprehensive set... < Topors-201protein coding < Ndufb6-201protein coding < Ndufb6-202protein coding Regulatory Build 40.265Mb 40.270Mb 40.275Mb 40.280Mb 40.285Mb Reverse strand 28.83 kb Regulation Legend CTCF Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000095128 < Ndufb6-201protein coding Reverse strand 8.83 kb ENSMUSP00000092... Transmembrane heli... PDB-ENSP mappings Low complexity (Seg) Pfam NADH dehydrogenase 1, beta subcomplex, subunit 6 PANTHER NADH dehydrogenase 1, beta subcomplex, subunit 6 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 128 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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