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Mouse Cox6c Conditional Knockout Project (CRISPR/Cas9)

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Mouse Cox6c Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Cox6c Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Cox6c conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cox6c gene (NCBI Reference Sequence: NM_053071 ; Ensembl: ENSMUSG00000014313 ) is located on Mouse chromosome 15. 4 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000014457). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cox6c gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-97E5 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 starts from about 100% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 840 bp, and the size of intron 2 for 3'-loxP site insertion: 1741 bp. The size of effective cKO region: ~617 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 4 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Cox6c 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. 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7117bp) | A(23.94% 1704) | C(22.03% 1568) | T(31.56% 2246) | G(22.47% 1599) 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 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% chr15 - 35937522 35940521 3000 browser details YourSeq 322 215 772 3000 90.9% chr10 - 3333984 3660651 326668 browser details YourSeq 280 168 624 3000 90.2% chr5 - 107648421 108033572 385152 browser details YourSeq 280 313 770 3000 88.3% chr11 + 22350609 22351099 491 browser details YourSeq 274 315 772 3000 89.0% chr3 - 132952417 132952883 467 browser details YourSeq 272 321 775 3000 87.2% chr11 - 62754681 62755164 484 browser details YourSeq 268 307 790 3000 90.9% chr5 + 103157032 103157547 516 browser details YourSeq 259 317 758 3000 88.8% chr4 - 48820969 48821431 463 browser details YourSeq 257 307 772 3000 86.7% chr17 + 69317070 69317590 521 browser details YourSeq 254 318 769 3000 89.0% chr17 - 75773809 75774336 528 browser details YourSeq 253 315 772 3000 86.2% chr2 + 51942778 51943234 457 browser details YourSeq 248 305 772 3000 84.9% chr19 + 6611796 6612240 445 browser details YourSeq 248 315 757 3000 89.5% chr18 + 84895558 84896019 462 browser details YourSeq 247 307 776 3000 84.6% chr6 - 72057513 72057966 454 browser details YourSeq 242 307 772 3000 84.2% chr9 + 48959794 48960215 422 browser details YourSeq 241 315 775 3000 85.1% chr17 + 49341725 49342170 446 browser details YourSeq 239 301 772 3000 82.5% chrX - 150474544 150475000 457 browser details YourSeq 236 307 749 3000 88.4% chr8 - 45158251 45158720 470 browser details YourSeq 236 315 699 3000 89.1% chr6 + 55574717 55575131 415 browser details YourSeq 229 295 772 3000 89.7% chr14 + 37331092 37331647 556 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% chr15 - 35933905 35936904 3000 browser details YourSeq 314 1981 2593 3000 90.6% chr17 + 65240191 65240744 554 browser details YourSeq 266 1982 2593 3000 90.9% chr1 + 95506959 95507771 813 browser details YourSeq 265 1980 2323 3000 92.7% chr13 - 60555359 60555719 361 browser details YourSeq 261 1982 2328 3000 91.5% chr5 - 9522110 9522473 364 browser details YourSeq 255 1982 2300 3000 93.6% chr17 + 47012867 47013201 335 browser details YourSeq 255 1982 2300 3000 93.6% chr15 + 91781926 91782260 335 browser details YourSeq 254 1981 2291 3000 94.5% chr13 - 67116017 67116357 341 browser details YourSeq 254 1982 2303 3000 92.6% chr13 + 67734604 67734934 331 browser details YourSeq 252 1988 2302 3000 93.5% chr11 + 47737798 47738127 330 browser details YourSeq 251 1982 2303 3000 92.0% chrX + 53980814 53981151 338 browser details YourSeq 250 1982 2322 3000 88.9% chr7 - 53937982 53938335 354 browser details YourSeq 248 1982 2305 3000 92.5% chr16 - 36110720 36111050 331 browser details YourSeq 247 1981 2303 3000 92.2% chr3 - 48530961 48531287 327 browser details YourSeq 247 1980 2314 3000 90.8% chr10 - 23256416 23256772 357 browser details YourSeq 246 1981 2291 3000 92.5% chr7 - 104013346 104013666 321 browser details YourSeq 245 1982 2303 3000 92.5% chr5 - 110075441 110075997 557 browser details YourSeq 245 1981 2305 3000 93.6% chr17 - 92428690 92429021 332 browser details YourSeq 243 1980 2580 3000 89.2% chr3 - 62589692 62590267 576 browser details YourSeq 240 1982 2323 3000 89.3% chr17 - 61917010 61917368 359 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Cox6c cytochrome c oxidase subunit 6C [ Mus musculus (house mouse) ] Gene ID: 12864, updated on 12-Aug-2019 Gene summary Official Symbol Cox6c provided by MGI Official Full Name cytochrome c oxidase subunit 6C provided by MGI Primary source MGI:MGI:104614 See related Ensembl:ENSMUSG00000014313 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 Ubiquitous expression in heart adult (RPKM 387.4), bladder adult (RPKM 339.2) and 28 other tissues See more Orthologs human all Genomic context Location: 15; 15 B3.1 See Cox6c in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (35931976..35938246, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (35861731..35868001, complement) Chromosome 15 - NC_000081.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Cox6c ENSMUSG00000014313 Description cytochrome c oxidase subunit 6C [Source:MGI Symbol;Acc:MGI:104614] Location Chromosome 15: 35,925,886-35,938,246 reverse strand. GRCm38:CM001008.2 About this gene This gene has 6 transcripts (splice variants), 89 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cox6c- ENSMUST00000153512.1 738 76aa ENSMUSP00000123609.1 Protein coding CCDS27424 Q9CPQ1 TSL:2 203 GENCODE basic APPRIS P1 Cox6c- ENSMUST00000156915.2 606 76aa ENSMUSP00000122391.2 Protein coding CCDS27424 Q9CPQ1 TSL:5 205 GENCODE basic APPRIS P1 Cox6c- ENSMUST00000014457.14 503 76aa ENSMUSP00000014457.8 Protein coding CCDS27424 Q9CPQ1 TSL:1 201 GENCODE basic APPRIS P1 Cox6c- ENSMUST00000155638.7 648 10aa ENSMUSP00000121106.1 Nonsense mediated - A0A0G2JDA6 CDS 5' 204 decay incomplete TSL:3 Cox6c- ENSMUST00000127999.1 3038 No - Retained intron - - TSL:1 202 protein Cox6c- ENSMUST00000227231.1 260 No - lncRNA - - - 206 protein Page 6 of 8 https://www.alphaknockout.com 32.36 kb Forward strand 35.92Mb 35.93Mb 35.94Mb Genes Vps13b-201 >protein coding (Comprehensive set... Contigs < AC157521.2 Genes (Comprehensive set... < Cox6c-204nonsense mediated decay < Cox6c-206lncRNA < Cox6c-201protein coding < Cox6c-203protein coding < Cox6c-202retained intron < Cox6c-205protein coding Regulatory Build 35.92Mb 35.93Mb 35.94Mb Reverse strand 32.36 kb Regulation Legend CTCF Enhancer 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: ENSMUST00000014457 < Cox6c-201protein coding Reverse strand 6.27 kb ENSMUSP00000014... Transmembrane heli... Superfamily Mitochondrial cytochrome c oxidase subunit VIc/VIIs superfamily Pfam Mitochondrial cytochrome c oxidase subunit VIc/VIIs PANTHER PTHR12916:SF0 PTHR12916 Gene3D Mitochondrial cytochrome c oxidase subunit VIc/VIIs superfamily CDD Cytochrome c oxidase subunit 6C All sequence SNPs/i... Sequence variants (dbSNP and all other sources) S Variant Legend synonymous variant Scale bar 0 8 16 24 32 40 48 56 64 76 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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