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Mouse Acox1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Acox1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Acox1 gene (NCBI Reference Sequence: NM_015729 ; Ensembl: ENSMUSG00000020777 ) is located on Mouse chromosome 11. 14 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000066587). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Acox1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-142K17 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 targeted mutation that inactivates the gene show growth retardation, infertility, excess very long chain fatty acids in the blood, and progressive liver disease, including hepatomegaly, and hepatic adenomas and carcinomas.

Exon 3 starts from about 13.62% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 14597 bp, and the size of intron 4 for 3'-loxP site insertion: 850 bp. The size of effective cKO region: ~2212 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 3 4 5 6 7 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Acox1 Homology arm 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(8712bp) | A(25.07% 2184) | C(22.96% 2000) | T(26.86% 2340) | G(25.11% 2188)

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% chr11 - 116183875 116186874 3000 browser details YourSeq 116 1710 1898 3000 82.9% chr15 + 75649284 75649467 184 browser details YourSeq 108 1710 1897 3000 77.8% chr2 + 102179194 102179374 181 browser details YourSeq 104 1721 1897 3000 78.7% chr9 - 64013957 64014129 173 browser details YourSeq 104 1710 1876 3000 88.3% chr5 + 123904685 123904854 170 browser details YourSeq 104 1710 1876 3000 88.3% chr15 + 76641627 76641793 167 browser details YourSeq 101 1729 1894 3000 86.3% chr11 + 79171506 79171677 172 browser details YourSeq 100 1719 1875 3000 82.2% chr18 + 34573164 34573318 155 browser details YourSeq 99 1710 1918 3000 85.9% chr9 - 109520025 109555081 35057 browser details YourSeq 97 1729 1901 3000 81.1% chr5 - 147915950 147916108 159 browser details YourSeq 96 1719 1910 3000 84.5% chr9 - 72962918 72963102 185 browser details YourSeq 96 1710 1841 3000 84.8% chr7 - 6865355 6865483 129 browser details YourSeq 96 1710 1906 3000 90.8% chr4 - 62644878 62645075 198 browser details YourSeq 93 1723 1880 3000 81.3% chr4 + 118299148 118299300 153 browser details YourSeq 91 1710 1856 3000 83.0% chr11 + 106958921 106959065 145 browser details YourSeq 89 1730 1897 3000 80.4% chr11 - 105096207 105096343 137 browser details YourSeq 89 1723 1858 3000 88.3% chr6 + 85425568 85426093 526 browser details YourSeq 88 1719 1875 3000 89.3% chr11 + 97721198 97721421 224 browser details YourSeq 88 1710 1838 3000 90.1% chr11 + 96360782 96360913 132 browser details YourSeq 87 1710 1880 3000 87.0% chr11 - 107018444 107018621 178

Note: The 3000 bp section upstream of Exon 3 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% chr11 - 116178663 116181662 3000 browser details YourSeq 352 25 389 3000 97.8% chr11 - 116181123 116181486 364 browser details YourSeq 350 177 538 3000 98.7% chr11 - 116181276 116181638 363 browser details YourSeq 301 25 338 3000 97.5% chr11 - 116181124 116181436 313 browser details YourSeq 217 765 2615 3000 93.3% chr2 - 28439230 28697472 258243 browser details YourSeq 215 754 2545 3000 96.2% chr1 + 86228998 86449711 220714 browser details YourSeq 181 774 1069 3000 83.4% chr14 - 47665970 47666267 298 browser details YourSeq 157 779 1057 3000 91.7% chrX - 61271821 61687783 415963 browser details YourSeq 156 778 1081 3000 92.9% chr9 + 88457447 88457914 468 browser details YourSeq 149 771 933 3000 94.5% chr4 + 139354467 139354628 162 browser details YourSeq 149 756 931 3000 94.2% chr2 + 103520170 103520359 190 browser details YourSeq 148 771 931 3000 96.3% chr5 - 115829163 115829326 164 browser details YourSeq 148 774 1054 3000 89.4% chr7 + 127653074 127653654 581 browser details YourSeq 147 754 929 3000 92.3% chr8 - 75007712 75007886 175 browser details YourSeq 147 751 929 3000 91.0% chr12 - 83572413 83572583 171 browser details YourSeq 147 771 1055 3000 94.1% chr13 + 73156145 73156431 287 browser details YourSeq 145 771 930 3000 95.7% chr3 - 121305690 121305901 212 browser details YourSeq 143 771 931 3000 95.6% chr7 - 68267959 68268146 188 browser details YourSeq 143 774 931 3000 95.6% chr13 - 63195980 63196138 159 browser details YourSeq 143 772 932 3000 92.6% chr12 - 23659946 23660094 149

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Acox1 acyl-Coenzyme A oxidase 1, palmitoyl [ Mus musculus (house mouse) ] Gene ID: 11430, updated on 10-Oct-2019

Gene summary

Official Symbol Acox1 provided by MGI Official Full Name acyl-Coenzyme A oxidase 1, palmitoyl provided by MGI Primary source MGI:MGI:1330812 See related Ensembl:ENSMUSG00000020777 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as AOX; Acox; Paox; D130055E20Rik Summary This gene encodes a member of the acyl-coenzyme A oxidase family. The encoded protein is localized to peroxisomes and Expression is the first enzyme of the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-coenzyme A to 2-trans- enoyl-coenzyme A. Disruption of this gene results in microvesicular steatohepatitis, spontaneous peroxisome proliferation, and the eventual development of hepatocellular carcinomas. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Dec 2012] Orthologs Broad expression in liver adult (RPKM 251.9), liver E18 (RPKM 159.5) and 21 other tissues See more human all

Genomic context

Location: 11; 11 E2 See Acox1 in Genome Data Viewer

Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (116171883..116199045, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (116033202..116060359, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 7 transcripts

Gene: Acox1 ENSMUSG00000020777

Description acyl-Coenzyme A oxidase 1, palmitoyl [Source:MGI Symbol;Acc:MGI:1330812] Gene Synonyms AOX, Acyl-CoA oxidase, D130055E20Rik Location Chromosome 11: 116,171,888-116,199,045 reverse strand. GRCm38:CM001004.2 About this gene This gene has 7 transcripts (splice variants), 214 orthologues, 15 paralogues, is a member of 1 Ensembl protein family and is associated with 24 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Acox1-201 ENSMUST00000066587.11 3987 661aa ENSMUSP00000063325.5 Protein coding CCDS25660 Q9R0H0 TSL:1 GENCODE basic APPRIS P3

Acox1-202 ENSMUST00000072948.10 3743 661aa ENSMUSP00000072717.4 Protein coding CCDS70354 Q9R0H0 TSL:1 GENCODE basic APPRIS ALT1

Acox1-206 ENSMUST00000148601.1 3538 625aa ENSMUSP00000122185.1 Protein coding - A2A848 CDS 5' incomplete TSL:2

Acox1-205 ENSMUST00000130229.7 3292 No protein - lncRNA - - TSL:1

Acox1-207 ENSMUST00000150549.7 954 No protein - lncRNA - - TSL:2

Acox1-203 ENSMUST00000124684.1 841 No protein - lncRNA - - TSL:2

Acox1-204 ENSMUST00000128793.1 798 No protein - lncRNA - - TSL:3

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47.16 kb Forward strand 116.17Mb 116.18Mb 116.19Mb 116.20Mb Genes Ten1-201 >protein coding (Comprehensive set...

Ten1-202 >retained intron

Ten1-204 >lncRNA

Contigs AL607108.17 > AL669925.8 > Genes (Comprehensive set... < Fbf1-201protein coding < Acox1-205lncRNA < Acox1-203lncRNA

< Fbf1-208lncRNA < Acox1-206protein coding

< Fbf1-202protein coding < Acox1-202protein coding

< Fbf1-203protein coding < Acox1-201protein coding

< Fbf1-206lncRNA < Acox1-207lncRNA

< Fbf1-211lncRNA < Acox1-204lncRNA

< Gm26413-202scaRNA

< Gm26413-201scaRNA

< Fbf1-209protein coding

< Fbf1-207retained intron

Regulatory Build

116.17Mb 116.18Mb 116.19Mb 116.20Mb Reverse strand 47.16 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: ENSMUST00000066587

< Acox1-201protein coding

Reverse strand 27.16 kb

ENSMUSP00000063... Superfamily Acyl-CoA dehydrogenase/oxidase, N-terminal and middle domain superfamily

Acyl-CoA dehydrogenase-like, C-terminal Pfam Acyl-coenzyme A oxidase, N-terminal Acyl-CoA oxidase, C-terminal

Acyl-CoA oxidase/dehydrogenase, central domain PIRSF Acyl-CoA oxidase PANTHER PTHR10909:SF250

PTHR10909 Gene3D Acyl-CoA dehydrogenase/oxidase, N-terminal domain superfamily

2.40.110.10 1.20.140.10 CDD Peroxisomal acyl-coenzyme A oxidase

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 661

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

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