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Mouse Cox4i2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cox4i2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cox4i2 (NCBI Reference Sequence: NM_053091 ; Ensembl: ENSMUSG00000009876 ) is located on Mouse 2. 5 are identified, with the ATG start codon in 2 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000010020). 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 Cox4i2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-479O11 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 null display inflammation and decreased airway responsiveness. Females show decreased lean body mass and improved glucose tolerance.

Exon 2 starts from about 100% 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: 930 bp, and the size of intron 3 for 3'-loxP site insertion: 3463 bp. The size of effective cKO region: ~2520 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 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Cox4i2 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. 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(9020bp) | A(24.47% 2207) | C(25.52% 2302) | T(25.13% 2267) | G(24.88% 2244)

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.

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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% chr2 + 152751906 152754905 3000 browser details YourSeq 161 539 856 3000 91.8% chr9 - 115224054 115224591 538 browser details YourSeq 151 536 814 3000 84.7% chr16 + 34832828 34833043 216 browser details YourSeq 148 537 717 3000 93.6% chr1 + 162604698 162605075 378 browser details YourSeq 145 537 816 3000 88.2% chr5 + 108039172 108039447 276 browser details YourSeq 144 536 698 3000 91.9% chr14 + 15289783 15289941 159 browser details YourSeq 141 535 698 3000 93.8% chr10 + 57505416 57505583 168 browser details YourSeq 140 551 816 3000 87.9% chr11 + 97076878 97077134 257 browser details YourSeq 140 535 698 3000 92.9% chr10 + 117720559 117720719 161 browser details YourSeq 140 541 808 3000 90.3% chr10 + 43424860 43521806 96947 browser details YourSeq 138 535 698 3000 91.3% chr1 - 135503230 135503391 162 browser details YourSeq 137 549 1143 3000 80.3% chr2 + 157080969 157081276 308 browser details YourSeq 136 535 693 3000 90.4% chr3 - 154186277 154186431 155 browser details YourSeq 135 535 698 3000 89.6% chr1 + 60210877 60211038 162 browser details YourSeq 133 535 697 3000 91.4% chr4 + 119558404 119558571 168 browser details YourSeq 133 535 698 3000 88.2% chr10 + 21950645 21950804 160 browser details YourSeq 133 535 698 3000 88.2% chr1 + 92433164 92433323 160 browser details YourSeq 133 532 694 3000 89.4% chr1 + 34850956 34851109 154 browser details YourSeq 132 547 698 3000 94.6% chr11 - 53147242 53147395 154 browser details YourSeq 132 535 693 3000 89.7% chr1 - 63117558 63117713 156

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% chr2 + 152757426 152760425 3000 browser details YourSeq 146 549 720 3000 93.1% chr17 - 25798605 25798802 198 browser details YourSeq 143 408 720 3000 88.8% chrX + 164239854 164240435 582 browser details YourSeq 141 547 736 3000 87.7% chr11 + 57823338 57823525 188 browser details YourSeq 140 550 720 3000 92.3% chr7 - 114119571 114119749 179 browser details YourSeq 140 552 720 3000 89.6% chr15 + 99312252 99312414 163 browser details YourSeq 139 563 742 3000 89.8% chr17 - 29424873 29425049 177 browser details YourSeq 139 550 721 3000 92.2% chr15 + 101247709 101247887 179 browser details YourSeq 138 564 737 3000 92.2% chr12 - 111277738 111277926 189 browser details YourSeq 138 552 728 3000 90.2% chr10 - 117196342 117196526 185 browser details YourSeq 138 540 733 3000 90.2% chr5 + 115078096 115078308 213 browser details YourSeq 137 553 720 3000 91.1% chr2 - 30373489 30373663 175 browser details YourSeq 137 553 719 3000 92.1% chr12 - 59686012 59686184 173 browser details YourSeq 137 539 720 3000 89.8% chr11 + 98689480 98689683 204 browser details YourSeq 137 563 720 3000 93.7% chr10 + 77655110 77655275 166 browser details YourSeq 137 550 720 3000 92.1% chr10 + 22634331 22634503 173 browser details YourSeq 137 557 720 3000 92.7% chr1 + 180701855 180702038 184 browser details YourSeq 136 563 720 3000 93.6% chr1 + 133837487 133837651 165 browser details YourSeq 135 553 720 3000 92.6% chr9 - 59465146 59465319 174 browser details YourSeq 135 552 720 3000 93.0% chr6 - 51596877 51597061 185

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 information: Cox4i2 subunit 4I2 [ Mus musculus (house mouse) ] Gene ID: 84682, updated on 13-Aug-2019

Gene summary

Official Symbol Cox4i2 provided by MGI Official Full Name cytochrome c oxidase subunit 4I2 provided by MGI Primary source MGI:MGI:2135755 See related Ensembl:ENSMUSG00000009876 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 Cox4b; CoxIV-2 Expression Biased expression in lung adult (RPKM 10.6), limb E14.5 (RPKM 2.1) and 8 other tissuesS ee more Orthologs human all

Genomic context

Location: 2; 2 H1 See Cox4i2 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (152753916..152765039)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (152579909..152590773)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 2 transcripts

Gene: Cox4i2 ENSMUSG00000009876

Description cytochrome c oxidase subunit 4I2 [Source:MGI Symbol;Acc:MGI:2135755] Gene Synonyms Cox IV-2, Cox4b Location Chromosome 2: 152,754,173-152,765,037 forward strand. GRCm38:CM000995.2 About this gene This gene has 2 transcripts (splice variants), 169 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cox4i2-202 ENSMUST00000109821.1 733 172aa ENSMUSP00000105446.1 Protein coding CCDS16898 Q91W29 TSL:1 GENCODE basic APPRIS P1

Cox4i2-201 ENSMUST00000010020.11 727 172aa ENSMUSP00000010020.5 Protein coding CCDS16898 Q91W29 TSL:1 GENCODE basic APPRIS P1

30.86 kb Forward strand 152.75Mb 152.76Mb 152.77Mb (Comprehensive set... Gm14162-201 >processed pseudogene Cox4i2-201 >protein coding

Cox4i2-202 >protein coding

Contigs < AL731857.9 Genes < Gm23802-201miRNA (Comprehensive set...

Regulatory Build

152.75Mb 152.76Mb 152.77Mb Reverse strand 30.86 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

pseudogene RNA gene

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Transcript: ENSMUST00000010020

10.87 kb Forward strand

Cox4i2-201 >protein coding

ENSMUSP00000010... Transmembrane heli... Low complexity (Seg) Superfamily Cytochrome c oxidase subunit IV superfamily Prints Cytochrome c oxidase subunit IV Pfam Cytochrome c oxidase subunit IV family PANTHER PTHR10707:SF11

Cytochrome c oxidase subunit IV family Gene3D Cytochrome c oxidase subunit IV superfamily CDD Cytochrome c oxidase subunit IV family

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend

frameshift variant missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 172

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

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