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Mouse Podxl Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Podxl gene (NCBI Reference Sequence: NM_013723 ; Ensembl: ENSMUSG00000025608 ) is located on Mouse chromosome 6. 8 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000026698). 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 Podxl gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-334G5 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 knock-out allele exhibit neonatal lethality and severe kidney defects including absence of the podocyte slit diaphragm and foot processes and anuria. While a subset display edema and/or omphalocele, most mice appear normal at birth.

Exon 3 starts from about 43.07% 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: 1702 bp, and the size of intron 4 for 3'-loxP site insertion: 1109 bp. The size of effective cKO region: ~1054 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 3 4 5 6 7 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Podxl 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(7554bp) | A(23.4% 1768) | C(26.56% 2006) | T(25.91% 1957) | G(24.13% 1823)

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% chr6 - 31527006 31530005 3000 browser details YourSeq 147 1603 1869 3000 88.8% chr10 + 62894722 62895127 406 browser details YourSeq 119 1596 1760 3000 84.8% chr10 - 62790656 62790807 152 browser details YourSeq 118 1569 1844 3000 81.4% chr8 + 104575258 104575486 229 browser details YourSeq 116 1624 1989 3000 88.2% chr12 + 84713438 84713842 405 browser details YourSeq 116 1597 1760 3000 84.3% chr12 + 65431978 65432124 147 browser details YourSeq 115 1602 1760 3000 91.0% chr1 - 166337004 166337160 157 browser details YourSeq 115 1591 1755 3000 91.3% chr1 - 164871474 164871651 178 browser details YourSeq 114 1591 1760 3000 81.8% chr19 - 57235073 57235226 154 browser details YourSeq 114 1640 1847 3000 87.2% chr13 - 26619029 26619491 463 browser details YourSeq 114 1597 1760 3000 82.7% chr11 + 53307329 53307478 150 browser details YourSeq 111 1623 1767 3000 85.1% chr13 + 95759146 95759280 135 browser details YourSeq 108 1609 1760 3000 83.5% chr8 - 110596283 110596422 140 browser details YourSeq 107 1615 1760 3000 85.0% chr7 + 66892649 66892787 139 browser details YourSeq 106 1623 1767 3000 85.2% chr11 + 29227974 29228108 135 browser details YourSeq 103 1611 1760 3000 89.9% chr8 - 65057253 65057404 152 browser details YourSeq 103 1623 1756 3000 88.9% chr7 - 110232840 110232972 133 browser details YourSeq 103 1599 1760 3000 91.8% chr5 + 55856414 55856589 176 browser details YourSeq 103 1612 1760 3000 83.0% chr15 + 66353502 66353638 137 browser details YourSeq 102 1623 1767 3000 81.9% chr18 - 9948099 9948230 132

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% chr6 - 31522952 31525951 3000 browser details YourSeq 52 2027 2126 3000 86.2% chr8 - 95814729 95814858 130 browser details YourSeq 40 2029 2109 3000 83.4% chr8 - 81406216 81406297 82 browser details YourSeq 37 2083 2129 3000 85.4% chr18 + 38287050 38287093 44 browser details YourSeq 36 2091 2266 3000 84.4% chr3 - 108049978 108050395 418 browser details YourSeq 35 2086 2126 3000 92.7% chr17 - 56440691 56440731 41 browser details YourSeq 35 2053 2111 3000 92.7% chr15 - 97140799 97140886 88 browser details YourSeq 34 2054 2111 3000 79.4% chr19 - 38894915 38894972 58 browser details YourSeq 34 2027 2111 3000 89.5% chr16 - 13369114 13369197 84 browser details YourSeq 34 2053 2126 3000 87.0% chr1 + 123992922 123993029 108 browser details YourSeq 33 2078 2124 3000 94.6% chr17 - 71282361 71282407 47 browser details YourSeq 33 2056 2112 3000 79.0% chr1 + 9916137 9916193 57 browser details YourSeq 32 2092 2129 3000 92.2% chr4 - 98399931 98399968 38 browser details YourSeq 32 2092 2128 3000 94.6% chr16 - 8839525 8839594 70 browser details YourSeq 32 2027 2111 3000 86.9% chr1 - 122193828 122193911 84 browser details YourSeq 31 2054 2114 3000 91.0% chr9 + 118103897 118103956 60 browser details YourSeq 31 2078 2128 3000 77.0% chr6 + 58861796 58861841 46 browser details YourSeq 31 2059 2101 3000 86.1% chr11 + 116826241 116826283 43 browser details YourSeq 31 2053 2107 3000 78.2% chr11 + 64250128 64250182 55 browser details YourSeq 29 2093 2129 3000 89.2% chr8 + 83942103 83942139 37

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: Podxl podocalyxin-like [ Mus musculus (house mouse) ] Gene ID: 27205, updated on 22-Oct-2019

Gene summary

Official Symbol Podxl provided by MGI Official Full Name podocalyxin-like provided by MGI Primary source MGI:MGI:1351317 See related Ensembl:ENSMUSG00000025608 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 PC; Ly102; Pclp1; PCLP-1; Podxl1; AW121214 Expression Broad expression in lung adult (RPKM 100.7), kidney adult (RPKM 59.6) and 18 other tissues See more Orthologs human all

Genomic context

Location: 6 A3.3; 6 12.57 cM See Podxl in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (31519493..31563937, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (31469493..31513937, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 2 transcripts

Gene: Podxl ENSMUSG00000025608

Description podocalyxin-like [Source:MGI Symbol;Acc:MGI:1351317] Gene Synonyms Ly102, PC, Pclp1, Podxl1, podocalyxin Location Chromosome 6: 31,519,488-31,563,981 reverse strand. GRCm38:CM000999.2 About this gene This gene has 2 transcripts (splice variants), 155 orthologues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Podxl-201 ENSMUST00000026698.7 5379 503aa ENSMUSP00000026698.7 Protein coding CCDS19983 Q791G4 Q9R0M4 TSL:1 GENCODE basic APPRIS P1

Podxl-202 ENSMUST00000136877.1 344 No protein - lncRNA - - TSL:5

64.49 kb Forward strand

Genes Mkln1-201 >protein coding (Comprehensive set...

Contigs < AC153837.4 Genes (Comprehensive set... < Podxl-201protein coding

< Podxl-202lncRNA

Regulatory Build

Reverse strand 64.49 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000026698

< Podxl-201protein coding

Reverse strand 44.49 kb

ENSMUSP00000026... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Pfam CD34/Podocalyxin PIRSF Podocalyxin PANTHER Podocalyxin

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

Variant Legend inframe insertion missense variant protein altering variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 503

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

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