https://www.alphaknockout.com Mouse Cox7a1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Cox7a1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cox7a1 gene (NCBI Reference Sequence: NM_009944 ; Ensembl: ENSMUSG00000074218 ) is located on Mouse chromosome 7. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000098594). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cox7a1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-162F12 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: Mice homozygous for a knock-out allele exhibit some premature death and dilated cardiomyopathy. Exon 2 starts from about 6.67% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 839 bp, and the size of intron 3 for 3'-loxP site insertion: 552 bp. The size of effective cKO region: ~775 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Cox7a1 cKO region Exon of mouse Capns1 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(7275bp) | A(23.97% 1744) | C(24.26% 1765) | T(26.25% 1910) | G(25.51% 1856) Note: The sequence of homologous arms and cKO region 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 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% chr7 + 30181836 30184835 3000 browser details YourSeq 331 250 1033 3000 90.3% chr4 + 33113087 33113475 389 browser details YourSeq 331 249 1034 3000 89.7% chr1 + 29218292 29218942 651 browser details YourSeq 316 250 1012 3000 88.9% chr10 - 76010634 76010999 366 browser details YourSeq 312 267 633 3000 94.4% chr10 - 6865581 6866303 723 browser details YourSeq 288 250 869 3000 96.5% chr10 + 51531186 51534198 3013 browser details YourSeq 286 253 1023 3000 85.6% chr9 - 72242600 72243091 492 browser details YourSeq 283 266 618 3000 94.1% chr5 + 145676738 145852760 176023 browser details YourSeq 280 238 562 3000 94.7% chrX + 110255329 110257956 2628 browser details YourSeq 277 250 585 3000 93.7% chr1 - 117662896 117663635 740 browser details YourSeq 276 246 548 3000 95.8% chr10 - 35327337 35327640 304 browser details YourSeq 275 239 541 3000 95.8% chrX - 55789947 55790521 575 browser details YourSeq 275 280 624 3000 92.6% chrX - 37056462 37056962 501 browser details YourSeq 273 250 541 3000 97.0% chr5 + 3145050 3145342 293 browser details YourSeq 272 249 541 3000 96.6% chr14 - 102677714 102678007 294 browser details YourSeq 272 249 541 3000 96.6% chr12 - 45429137 45429430 294 browser details YourSeq 271 212 541 3000 94.8% chrX - 147220432 147220971 540 browser details YourSeq 271 243 551 3000 94.5% chr12 - 114926988 114927300 313 browser details YourSeq 271 265 607 3000 92.3% chr1 - 77134742 77135081 340 browser details YourSeq 270 250 542 3000 96.3% chr4 + 83783390 83783683 294 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% chr7 + 30185611 30188610 3000 browser details YourSeq 106 886 1142 3000 90.7% chr11 + 6884331 6884603 273 browser details YourSeq 103 854 1091 3000 89.3% chr1 + 187592703 187593001 299 browser details YourSeq 100 861 1170 3000 93.9% chr10 - 121889149 121889699 551 browser details YourSeq 97 907 1170 3000 85.9% chr1 - 166350829 166351068 240 browser details YourSeq 94 821 1089 3000 89.1% chr1 - 192195712 192195981 270 browser details YourSeq 84 900 1178 3000 91.1% chr10 + 40127673 40128328 656 browser details YourSeq 75 1033 1171 3000 83.2% chr10 + 9436847 9436955 109 browser details YourSeq 73 858 1171 3000 91.8% chr10 - 75476571 75793361 316791 browser details YourSeq 71 857 2294 3000 91.8% chr11 - 120487774 120759120 271347 browser details YourSeq 70 854 1167 3000 72.5% chr10 - 113239604 113239712 109 browser details YourSeq 68 920 1135 3000 78.3% chr1 - 191303463 191303622 160 browser details YourSeq 67 999 1168 3000 94.8% chr1 - 7118907 7119196 290 browser details YourSeq 62 2179 2298 3000 93.2% chr10 - 62387445 62387613 169 browser details YourSeq 62 919 1143 3000 72.1% chr1 - 67977587 67977664 78 browser details YourSeq 62 1069 1170 3000 88.8% chr11 + 16558499 16558598 100 browser details YourSeq 54 1069 1217 3000 93.8% chr10 + 41945212 41945873 662 browser details YourSeq 53 2270 2478 3000 72.4% chr11 + 75688501 75688662 162 browser details YourSeq 52 2273 2546 3000 96.5% chr8 - 106692832 106693166 335 browser details YourSeq 50 1177 1270 3000 77.8% chr12 - 3974379 3974546 168 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Cox7a1 cytochrome c oxidase subunit 7A1 [ Mus musculus (house mouse) ] Gene ID: 12865, updated on 12-Aug-2019 Gene summary Official Symbol Cox7a1 provided by MGI Official Full Name cytochrome c oxidase subunit 7A1 provided by MGI Primary source MGI:MGI:1316714 See related Ensembl:ENSMUSG00000074218 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 COX7A; COX7AH; COX7AM Expression Biased expression in heart adult (RPKM 444.7), large intestine adult (RPKM 31.1) and 2 other tissues See more Orthologs human all Genomic context Location: 7 B1; 7 17.31 cM See Cox7a1 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (30184171..30186030) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (30969190..30971049) Chromosome 7 - NC_000073.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Cox7a1 ENSMUSG00000074218 Description cytochrome c oxidase subunit 7A1 [Source:MGI Symbol;Acc:MGI:1316714] Gene Synonyms COX7A, COX7AH, COX7AM Location Chromosome 7: 30,184,144-30,186,078 forward strand. GRCm38:CM001000.2 About this gene This gene has 2 transcripts (splice variants), 95 orthologues, 2 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 Cox7a1-201 ENSMUST00000098594.3 393 80aa ENSMUSP00000096193.2 Protein coding CCDS21081 P56392 Q792A4 TSL:1 GENCODE basic APPRIS P1 Cox7a1-202 ENSMUST00000208441.1 485 89aa ENSMUSP00000146960.1 Protein coding - A0A140LIU4 TSL:2 GENCODE basic 21.93 kb Forward strand 30.175Mb 30.180Mb 30.185Mb 30.190Mb 30.195Mb Genes (Comprehensive set... Gm5113-201 >lncRNA Cox7a1-201 >protein coding Gm26810-202 >lncRNA Gm5113-202 >transcribed unprocessed pseudogene Cox7a1-202 >protein coding Contigs AC164703.3 > Genes < Capns1-208retained intron < Capns1-206retained intron (Comprehensive set... < Capns1-201protein coding < Capns1-203protein coding < Capns1-202protein coding < Capns1-207retained intron < Capns1-204lncRNA < Capns1-205retained intron Regulatory Build 30.175Mb 30.180Mb 30.185Mb 30.190Mb 30.195Mb Reverse strand 21.93 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000098594 1.89 kb Forward strand Cox7a1-201 >protein coding ENSMUSP00000096... Transmembrane heli... Superfamily Cytochrome c oxidase, subunit VIIa superfamily Pfam Cytochrome c oxidase subunit VII PANTHER PTHR10510:SF5 Cytochrome c oxidase subunit VIIa, metazoa Gene3D Cytochrome c oxidase, subunit VIIa superfamily CDD Cytochrome c oxidase subunit VIIa, metazoa All sequence SNPs/i... Sequence variants (dbSNP and all other sources) R R Variant Legend synonymous variant Scale bar 0 8 16 24 32 40 48 56 64 72 80 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.