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Mouse Pex13 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Pex13 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Pex13 gene (NCBI Reference Sequence: NM_023651 ; Ensembl: ENSMUSG00000020283 ) is located on Mouse chromosome 11. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000020523). 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 Pex13 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-297N11 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: Targeted disruption of this gene results in intrauterine growth retardation, hypotonia, aphagia, abnormal lamination of the cerebral cortex associated with a neuronal migration defect, liver steatosis, delayed differentiation of renal glomeruli, impairedperoxisome metabolism, and neonatal death.
Exon 2 starts from about 8.15% 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: 9616 bp, and the size of intron 2 for 3'-loxP site insertion: 4380 bp. The size of effective cKO region: ~1195 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele gRNA region 5' gRNA region 3'
1 2 4 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Pex13 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. 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(7695bp) | A(32.81% 2525) | C(17.15% 1320) | T(28.69% 2208) | G(21.34% 1642)
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 - 23656381 23659380 3000 browser details YourSeq 49 370 429 3000 91.6% chr1 + 74424854 74424916 63 browser details YourSeq 48 380 434 3000 88.3% chr7 + 109495847 109495898 52 browser details YourSeq 48 379 440 3000 90.4% chr12 + 26420665 26421014 350 browser details YourSeq 46 375 429 3000 92.8% chr1 + 71136938 71136995 58 browser details YourSeq 45 383 476 3000 94.2% chr5 - 134685824 134685923 100 browser details YourSeq 45 382 476 3000 74.2% chr1 + 163903336 163903431 96 browser details YourSeq 44 380 429 3000 94.0% chr8 + 128650918 128650967 50 browser details YourSeq 44 381 430 3000 94.0% chr8 + 26091761 26091810 50 browser details YourSeq 43 379 490 3000 70.1% chr12 - 8818614 8818734 121 browser details YourSeq 42 413 476 3000 82.9% chr1 - 118824590 118824653 64 browser details YourSeq 42 379 424 3000 95.7% chr7 + 34252899 34252944 46 browser details YourSeq 41 396 477 3000 75.4% chr19 - 43873705 43873789 85 browser details YourSeq 41 383 429 3000 93.7% chr17 - 30890954 30891000 47 browser details YourSeq 41 405 477 3000 78.1% chr13 - 97737141 97737213 73 browser details YourSeq 41 380 424 3000 95.6% chrX + 52278886 52278930 45 browser details YourSeq 41 404 490 3000 74.7% chr11 + 114134156 114134256 101 browser details YourSeq 40 395 476 3000 89.2% chr14 - 52060268 52060348 81 browser details YourSeq 40 379 424 3000 88.9% chr5 + 136488291 136488335 45 browser details YourSeq 40 379 426 3000 87.3% chr1 + 172453451 172453497 47
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% chr11 - 23652186 23655185 3000 browser details YourSeq 196 2438 2942 3000 86.0% chr2 + 38329603 38648689 319087 browser details YourSeq 193 2438 2965 3000 92.6% chr7 + 79650303 79672153 21851 browser details YourSeq 150 2438 2618 3000 91.8% chr15 - 62373066 62373250 185 browser details YourSeq 149 2445 2688 3000 87.0% chr2 + 121097594 121098014 421 browser details YourSeq 147 2216 2618 3000 79.3% chr2 + 154543089 154543315 227 browser details YourSeq 146 2438 2620 3000 90.2% chr4 + 43472392 43472596 205 browser details YourSeq 145 2438 2616 3000 91.5% chr2 + 65294968 65295152 185 browser details YourSeq 144 2438 2618 3000 91.0% chr8 + 26113513 26113695 183 browser details YourSeq 142 2438 2617 3000 89.9% chr7 + 116403573 116403755 183 browser details YourSeq 141 2438 2618 3000 89.0% chr10 - 70480955 70481135 181 browser details YourSeq 141 2438 2618 3000 89.5% chr1 - 134414029 134414213 185 browser details YourSeq 139 2438 2619 3000 88.4% chrX + 8113807 8113990 184 browser details YourSeq 138 2438 2641 3000 86.4% chr6 - 23456897 23457099 203 browser details YourSeq 137 2438 2606 3000 90.6% chr17 - 71488906 71489074 169 browser details YourSeq 136 2438 2690 3000 84.3% chr6 - 38790919 38791315 397 browser details YourSeq 135 2419 2616 3000 84.4% chrX + 73163924 73164125 202 browser details YourSeq 135 2443 2610 3000 90.5% chr3 + 100509018 100509189 172 browser details YourSeq 135 2443 2616 3000 90.5% chr10 + 41191267 41191443 177 browser details YourSeq 134 2419 2606 3000 85.7% chrX - 33061505 33061692 188
Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Pex13 peroxisomal biogenesis factor 13 [ Mus musculus (house mouse) ] Gene ID: 72129, updated on 12-Aug-2019
Gene summary
Official Symbol Pex13 provided by MGI Official Full Name peroxisomal biogenesis factor 13 provided by MGI Primary source MGI:MGI:1919379 See related Ensembl:ENSMUSG00000020283 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 2610008O20Rik Expression Ubiquitous expression in testis adult (RPKM 13.1), adrenal adult (RPKM 11.3) and 28 other tissues See more Orthologs human all
Genomic context
Location: 11; 11 A3.2 See Pex13 in Genome Data Viewer
Exon count: 4
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (23646843..23665883, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (23546479..23565935, complement)
Chromosome 11 - NC_000077.6
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Transcript information: This gene has 4 transcripts
Gene: Pex13 ENSMUSG00000020283
Description peroxisomal biogenesis factor 13 [Source:MGI Symbol;Acc:MGI:1919379] Gene Synonyms 2610008O20Rik Location Chromosome 11: 23,646,479-23,665,959 reverse strand. GRCm38:CM001004.2 About this gene This gene has 4 transcripts (splice variants), 192 orthologues, is a member of 1 Ensembl protein family and is associated with 19 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Pex13- ENSMUST00000020523.3 4146 405aa ENSMUSP00000020523.3 Protein coding CCDS24478 Q9D0K1 TSL:1 201 GENCODE basic APPRIS P1
Pex13- ENSMUST00000130811.1 367 40aa ENSMUSP00000115020.1 Nonsense mediated - D6RH41 TSL:3 203 decay
Pex13- ENSMUST00000124839.1 592 No - lncRNA - - TSL:3 202 protein
Pex13- ENSMUST00000146533.1 345 No - lncRNA - - TSL:3 204 protein
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39.48 kb Forward strand 23.64Mb 23.65Mb 23.66Mb 23.67Mb Genes Pus10-203 >protein coding (Comprehensive set...
Pus10-201 >protein coding
Pus10-202 >protein coding
Pus10-204 >retained intron
Pus10-207 >protein coding
Contigs AL672049.13 > Genes (Comprehensive set... < Pex13-201protein coding
< Pex13-203nonsense mediated decay
< Pex13-202lncRNA
< Pex13-204lncRNA
Regulatory Build
23.64Mb 23.65Mb 23.66Mb 23.67Mb Reverse strand 39.48 kb
Regulation Legend CTCF Enhancer 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: ENSMUST00000020523
< Pex13-201protein coding
Reverse strand 19.48 kb
ENSMUSP00000020... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Superfamily SH3-like domain superfamily SMART SH3 domain Prints SH3 domain Pfam Peroxin 13, N-terminal SH3 domain
PROSITE profiles SH3 domain PANTHER Peroxin 13
PTHR19332:SF5 Gene3D 2.30.30.40 CDD cd11864
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant synonymous variant
Scale bar 0 40 80 120 160 200 240 280 320 360 405
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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