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Mouse Pex16 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Pex16 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Pex16 gene (NCBI Reference Sequence: NM_145122 ; Ensembl: ENSMUSG00000027222 ) is located on Mouse chromosome 2. 11 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000028650). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pex16 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-316M14 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 4 starts from about 22.42% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 726 bp, and the size of intron 5 for 3'-loxP site insertion: 801 bp. The size of effective cKO region: ~862 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 5 6 7 8 9 11 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Pex16 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(7362bp) | A(21.42% 1577) | C(25.44% 1873) | T(24.68% 1817) | G(28.46% 2095)

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 + 92374181 92377180 3000 browser details YourSeq 145 2733 2992 3000 84.3% chr11 - 70523557 70523795 239 browser details YourSeq 137 2696 2874 3000 90.6% chr2 + 104468178 104468357 180 browser details YourSeq 135 2695 2878 3000 84.2% chr12 - 94896873 94897042 170 browser details YourSeq 133 2695 2878 3000 88.0% chr10 - 61572826 61573157 332 browser details YourSeq 132 2711 2879 3000 91.9% chr6 - 134422897 134423086 190 browser details YourSeq 130 2706 2875 3000 89.2% chr10 - 20346627 20346873 247 browser details YourSeq 129 2694 2856 3000 90.7% chr10 + 100482117 100482282 166 browser details YourSeq 128 2719 2878 3000 91.0% chr17 + 25264855 25265018 164 browser details YourSeq 121 2736 2878 3000 92.9% chr14 + 31078232 31078385 154 browser details YourSeq 121 2736 2884 3000 88.5% chr10 + 41470191 41470337 147 browser details YourSeq 120 2829 2992 3000 90.6% chr11 + 80175070 80591546 416477 browser details YourSeq 119 2736 2884 3000 87.8% chr13 + 9020811 9020957 147 browser details YourSeq 118 2733 2874 3000 91.6% chr6 - 19202575 19202716 142 browser details YourSeq 118 2742 2882 3000 92.2% chr3 + 89922049 89922209 161 browser details YourSeq 118 2733 2879 3000 90.5% chr16 + 70086831 70086978 148 browser details YourSeq 117 2742 2879 3000 92.8% chr16 - 40842452 40842592 141 browser details YourSeq 117 2733 2879 3000 88.4% chr16 - 3069525 3069670 146 browser details YourSeq 117 2735 2878 3000 91.5% chr12 - 11102114 11102258 145 browser details YourSeq 117 2733 2879 3000 88.4% chr19 + 3010067 3010212 146

Note: The 3000 bp section upstream of Exon 4 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 + 92378043 92381042 3000 browser details YourSeq 24 2969 2993 3000 100.0% chr1 + 91026446 91026478 33 browser details YourSeq 23 2961 2986 3000 96.0% chr14 - 24216586 24216611 26 browser details YourSeq 21 1645 1665 3000 100.0% chr1 - 42296686 42296706 21 browser details YourSeq 21 7 27 3000 100.0% chr1 + 20349567 20349587 21

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Pex16 peroxisomal biogenesis factor 16 [ Mus musculus (house mouse) ] Gene ID: 18633, updated on 12-Aug-2019

Gene summary

Official Symbol Pex16 provided by MGI Official Full Name peroxisomal biogenesis factor 16 provided by MGI Primary source MGI:MGI:1338829 See related Ensembl:ENSMUSG00000027222 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 Expression Ubiquitous expression in adrenal adult (RPKM 68.1), subcutaneous fat pad adult (RPKM 52.9) and 28 other tissues See Orthologs more human all

Genomic context

Location: 2; 2 E1 See Pex16 in Genome Data Viewer Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (92374676..92381220)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (92214833..92221377)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 4 transcripts

Gene: Pex16 ENSMUSG00000027222

Description peroxisomal biogenesis factor 16 [Source:MGI Symbol;Acc:MGI:1338829] Gene Synonyms peroxisome biogenesis factor 16 Location Chromosome 2: 92,374,676-92,381,217 forward strand. GRCm38:CM000995.2 About this gene This gene has 4 transcripts (splice variants), 193 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pex16-201 ENSMUST00000028650.8 1392 336aa ENSMUSP00000028650.8 Protein coding CCDS16444 Q91XC9 TSL:1 GENCODE basic APPRIS P1

Pex16-204 ENSMUST00000155891.7 1167 No protein - lncRNA - - TSL:1

Pex16-203 ENSMUST00000154669.7 852 No protein - lncRNA - - TSL:3

Pex16-202 ENSMUST00000148386.1 425 No protein - lncRNA - - TSL:3

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26.54 kb Forward strand 92.37Mb 92.38Mb 92.39Mb Genes (Comprehensive set... Pex16-204 >lncRNA 1700029I15Rik-203 >retained intron

Pex16-201 >protein coding 1700029I15Rik-202 >protein coding

Pex16-203 >lncRNA 1700029I15Rik-201 >protein coding

Pex16-202 >lncRNA 1700029I15Rik-204 >nonsense mediated decay

Contigs AL731709.19 > Genes < Large2-201protein coding < Mapk8ip1-202protein coding (Comprehensive set...

< Large2-214protein coding < Mapk8ip1-201protein coding

< Large2-205retained intron < Mir7000-201miRNA

< Large2-207nonsense mediated decay < Mapk8ip1-203lncRNA

< Large2-210nonsense mediated decay

< Large2-203protein coding

< Large2-215protein coding

< Large2-202protein coding

< Large2-217retained intron< Large2-208retained intron

< Large2-209retained intron< Large2-211protein coding

< Large2-213retained intron

< Large2-216lncRNA

< Large2-212retained intron

< Large2-206retained intron

< Large2-204retained intron

Regulatory Build

92.37Mb 92.38Mb 92.39Mb Reverse strand 26.54 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000028650

5.91 kb Forward strand

Pex16-201 >protein coding

ENSMUSP00000028... Low complexity (Seg) Pfam Peroxisome membrane protein, Pex16 PANTHER PTHR13299:SF0

Peroxisome membrane protein, Pex16

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 336

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

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