https://www.alphaknockout.com Mouse Cox6a1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Cox6a1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cox6a1 gene (NCBI Reference Sequence: NM_007748 ; Ensembl: ENSMUSG00000041697 ) is located on Mouse chromosome 5. 3 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 3 (Transcript: ENSMUST00000040154). Exon 1~2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cox6a1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-322D18 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: Homozygous null mice exhibit impaired coordination, thinned sciatic nerves, neurogenic muscular changes and delayed motor nerve conduction velocity. Exon 1~2 covers 75.89% of the coding region. Start codon is in exon 1, and stop codon is in exon 3. The size of intron 2 for 3'-loxP site insertion: 2638 bp. The size of effective cKO region: ~646 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 A gRNA region T 5' G 3' 1 2 3 Targeting vector A T G Targeted allele A T G Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Cox6a1 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. 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(6886bp) | A(25.27% 1740) | C(23.66% 1629) | T(26.46% 1822) | G(24.62% 1695) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. 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% chr5 - 115348951 115351950 3000 browser details YourSeq 481 1159 2038 3000 93.3% chr16 + 56059841 56060687 847 browser details YourSeq 437 1197 2047 3000 93.5% chrX + 154415912 154416799 888 browser details YourSeq 419 1173 2047 3000 92.6% chr10 + 63311988 63312721 734 browser details YourSeq 403 1173 2047 3000 91.4% chr14 - 58188932 58189633 702 browser details YourSeq 400 1159 2045 3000 92.1% chr4 - 62334393 62335118 726 browser details YourSeq 390 1241 2047 3000 95.8% chr7 + 5417066 5417941 876 browser details YourSeq 383 1155 2047 3000 93.3% chr14 - 11233957 11234769 813 browser details YourSeq 380 1655 2049 3000 98.3% chr14 - 32416645 32417041 397 browser details YourSeq 380 1655 2047 3000 98.5% chr9 + 91792716 91793110 395 browser details YourSeq 378 1366 2047 3000 96.4% chr2 - 101050983 101051826 844 browser details YourSeq 378 1655 2047 3000 98.3% chr15 - 11632181 11632575 395 browser details YourSeq 375 1652 2047 3000 97.5% chr6 + 143014339 143014736 398 browser details YourSeq 374 1655 2047 3000 97.8% chr12 - 48374756 48375150 395 browser details YourSeq 374 1655 2047 3000 97.8% chr12 - 48376244 48376638 395 browser details YourSeq 373 1653 2048 3000 97.3% chr6 + 143015831 143016228 398 browser details YourSeq 373 1655 2057 3000 96.8% chr4 + 111103736 111104145 410 browser details YourSeq 373 1655 2048 3000 97.5% chr17 + 19830487 19830882 396 browser details YourSeq 372 1655 2047 3000 97.5% chr1 - 78301235 78301629 395 browser details YourSeq 372 1655 2047 3000 97.5% chr17 + 19828995 19829389 395 Note: The 3000 bp section upstream of Exon 1 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% chr5 - 115345315 115348314 3000 browser details YourSeq 278 1241 2174 3000 92.7% chr13 - 113009136 113475701 466566 browser details YourSeq 259 963 1609 3000 93.1% chr17 + 80219270 80330633 111364 browser details YourSeq 253 2387 2661 3000 94.5% chr19 + 3205988 3206259 272 browser details YourSeq 249 1250 1614 3000 92.5% chr5 - 29634240 29634802 563 browser details YourSeq 232 1264 1584 3000 89.4% chr10 - 91130348 91130649 302 browser details YourSeq 221 1319 1622 3000 90.5% chr4 + 129885917 129886226 310 browser details YourSeq 214 1305 1609 3000 90.9% chr1 - 63090945 63091491 547 browser details YourSeq 181 1233 1585 3000 85.5% chr8 + 111113887 111114213 327 browser details YourSeq 172 1392 1614 3000 91.8% chr4 - 134698271 134698871 601 browser details YourSeq 171 1359 1614 3000 87.1% chr19 - 44352276 44352497 222 browser details YourSeq 171 1346 1614 3000 89.1% chr4 + 116281615 116281961 347 browser details YourSeq 167 1244 1461 3000 88.9% chr12 + 52713794 52713985 192 browser details YourSeq 165 1258 1614 3000 89.2% chr14 - 31357593 31357941 349 browser details YourSeq 163 1330 1934 3000 93.1% chr9 + 104093646 104094284 639 browser details YourSeq 162 1303 1594 3000 93.2% chr11 - 50165566 50166346 781 browser details YourSeq 153 1241 1448 3000 86.6% chr5 - 116808478 116808656 179 browser details YourSeq 152 1264 1775 3000 86.2% chr10 + 36636472 36636911 440 browser details YourSeq 147 1319 1592 3000 89.8% chr16 + 18887015 18887567 553 browser details YourSeq 146 1452 1636 3000 93.5% chr9 - 58573408 58573598 191 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: Cox6a1 cytochrome c oxidase subunit 6A1 [ Mus musculus (house mouse) ] Gene ID: 12861, updated on 12-Aug-2019 Gene summary Official Symbol Cox6a1 provided by MGI Official Full Name cytochrome c oxidase subunit 6A1 provided by MGI Primary source MGI:MGI:103099 See related Ensembl:ENSMUSG00000041697 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 VIaL Expression Ubiquitous expression in duodenum adult (RPKM 2589.4), colon adult (RPKM 2186.7) and 28 other tissues See more Orthologs human all Genomic context Location: 5 F; 5 56.06 cM See Cox6a1 in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (115345623..115348958, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (115795663..115798964, complement) Chromosome 5 - NC_000071.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Cox6a1 ENSMUSG00000041697 Description cytochrome c oxidase subunit 6A1 [Source:MGI Symbol;Acc:MGI:103099] Gene Synonyms VIaL, subunit VIaL (liver-type) Location Chromosome 5: 115,345,642-115,348,981 reverse strand. GRCm38:CM000998.2 About this gene This gene has 2 transcripts (splice variants), 225 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cox6a1-201 ENSMUST00000040154.8 571 112aa ENSMUSP00000047661.8 Protein coding CCDS19590 Q9DCW5 TSL:1 GENCODE basic APPRIS P1 Cox6a1-202 ENSMUST00000137766.1 620 No protein - Retained intron - - TSL:2 23.34 kb Forward strand 115.340Mb 115.345Mb 115.350Mb 115.355Mb Genes Triap1-201 >protein coding Gm42903-201 >lncRNA (Comprehensive set... Contigs < AC117735.8 Genes (Comprehensive set... < Gatc-201protein coding < Cox6a1-201protein coding < 4930401G09Rik-201lncRNA < Cox6a1-202retained intron Regulatory Build 115.340Mb 115.345Mb 115.350Mb 115.355Mb Reverse strand 23.34 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000040154 < Cox6a1-201protein coding Reverse strand 3.34 kb ENSMUSP00000047... Transmembrane heli... Low complexity (Seg) Superfamily Cytochrome c oxidase, subunit VIa superfamily Pfam Cytochrome c oxidase, subunit VIa PROSITE patterns Cytochrome c oxidase, subunit VIa, conserved site PIRSF Cytochrome c oxidase, subunit VIa PANTHER Cytochrome c oxidase, subunit VIa PTHR11504:SF4 Gene3D Cytochrome c oxidase, subunit VIa superfamily CDD Cytochrome c oxidase, subunit VIa All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 10 20 30 40 50 60 70 80 90 100 112 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.