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Mouse Cox7a2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cox7a2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cox7a2 (NCBI Reference Sequence: NM_009945 ; Ensembl: ENSMUSG00000032330 ) is located on Mouse 9. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000034881). 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 Cox7a2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-124D16 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 7.63% 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: 1060 bp, and the size of intron 3 for 3'-loxP site insertion: 671 bp. The size of effective cKO region: ~2803 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Cox7a2 cKO region loxP site

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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(9303bp) | A(28.6% 2661) | C(18.36% 1708) | T(30.54% 2841) | G(22.5% 2093)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr9 - 79758830 79761829 3000 browser details YourSeq 177 625 803 3000 99.5% chr3 + 89155643 89155821 179 browser details YourSeq 167 625 805 3000 96.2% chr9 - 86669531 86669711 181 browser details YourSeq 162 625 823 3000 90.9% chr5 + 102012248 102012441 194 browser details YourSeq 157 625 968 3000 89.6% chr2 - 5633435 5634057 623 browser details YourSeq 156 625 803 3000 94.5% chr3 - 94840797 94840989 193 browser details YourSeq 156 625 818 3000 93.0% chr16 - 18844546 18844747 202 browser details YourSeq 153 623 819 3000 90.4% chr18 + 67220520 67220711 192 browser details YourSeq 152 625 801 3000 93.8% chr7 - 52755910 52756092 183 browser details YourSeq 152 626 801 3000 94.3% chr13 + 58533414 58533596 183 browser details YourSeq 151 625 802 3000 93.2% chr4 - 156114180 156114362 183 browser details YourSeq 151 626 801 3000 93.7% chr11 - 58076846 58077027 182 browser details YourSeq 151 625 802 3000 92.7% chr8 + 25796139 25796323 185 browser details YourSeq 151 625 803 3000 92.8% chr6 + 120455539 120455721 183 browser details YourSeq 151 625 802 3000 94.2% chr5 + 136189251 136189433 183 browser details YourSeq 151 625 802 3000 93.8% chr5 + 76117884 76118069 186 browser details YourSeq 150 625 805 3000 91.8% chr9 - 107620806 107620993 188 browser details YourSeq 150 625 805 3000 93.2% chrX + 162711105 162711291 187 browser details YourSeq 150 626 804 3000 93.7% chr4 + 11542507 11542694 188 browser details YourSeq 150 625 801 3000 93.7% chr3 + 33891069 33891252 184

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% chr9 - 79753027 79756026 3000 browser details YourSeq 182 428 645 3000 94.7% chr1 - 126708260 126708666 407 browser details YourSeq 170 421 620 3000 91.5% chr12 - 73236575 73236773 199 browser details YourSeq 170 421 616 3000 93.4% chr2 + 25543659 25543854 196 browser details YourSeq 48 536 609 3000 89.3% chr7 + 113491984 113492056 73 browser details YourSeq 23 1298 1330 3000 84.9% chr13 - 87222869 87222901 33 browser details YourSeq 21 683 709 3000 88.9% chr1 + 127080341 127080367 27

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cox7a2 subunit 7A2 [ Mus musculus (house mouse) ] Gene ID: 12866, updated on 12-Aug-2019

Gene summary

Official Symbol Cox7a2 provided by MGI Official Full Name cytochrome c oxidase subunit 7A2 provided by MGI Primary source MGI:MGI:1316715 See related Ensembl:ENSMUSG00000032330 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 COX7AL; Cox7a3; CoxVIIa-L Expression Ubiquitous expression in heart adult (RPKM 183.5), kidney adult (RPKM 165.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 9 E1; 9 43.82 cM See Cox7a2 in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (79755241..79771422, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (79603048..79607660, complement)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 3 transcripts

Gene: Cox7a2 ENSMUSG00000032330

Description cytochrome c oxidase subunit 7A2 [Source:MGI Symbol;Acc:MGI:1316715] Gene Synonyms COX7AL, Cox7a3 Location Chromosome 9: 79,755,361-79,759,878 reverse strand. GRCm38:CM001002.2 About this gene This gene has 3 transcripts (splice variants), 281 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cox7a2-201 ENSMUST00000034881.7 659 83aa ENSMUSP00000034881.6 Protein coding CCDS23366 P48771 TSL:1 GENCODE basic APPRIS P1

Cox7a2-202 ENSMUST00000215452.1 344 No protein - Retained intron - - TSL:2

Cox7a2-203 ENSMUST00000215933.1 477 No protein - lncRNA - - TSL:3

24.52 kb Forward strand 79.750Mb 79.755Mb 79.760Mb 79.765Mb Contigs AC140247.3 > (Comprehensive set... < Cox7a2-201protein coding < Tmem30a-202protein coding

< Cox7a2-203lncRNA < Tmem30a-201protein coding

< Cox7a2-202retained intron

Regulatory Build

79.750Mb 79.755Mb 79.760Mb 79.765Mb Reverse strand 24.52 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000034881

< Cox7a2-201protein coding

Reverse strand 4.52 kb

ENSMUSP00000034... Transmembrane heli... Superfamily Cytochrome c oxidase, subunit VIIa superfamily Pfam Cytochrome c oxidase subunit VII PANTHER Cytochrome c oxidase subunit VIIa, metazoa

PTHR10510:SF15 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

Variant Legend synonymous variant

Scale bar 0 8 16 24 32 40 48 56 64 72 83

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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