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Mouse Cldn3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cldn3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cldn3 (NCBI Reference Sequence: NM_009902 ; Ensembl: ENSMUSG00000070473 ) is located on Mouse 5. 1 exon is identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000094245). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cldn3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-357G21 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 allele are fertile with mutant males exhibiting normal spermatogenesis and fully functional Sertoli cell tight junctions.

Exon 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~1538 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region

Wildtype allele A T

5' G gRNA region 3'

1

Targeting vector A T G

Targeted allele A T G

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Cldn3 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(6657bp) | A(23.45% 1561) | C(24.47% 1629) | T(23.61% 1572) | G(28.47% 1895)

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% chr5 + 134983446 134986445 3000 browser details YourSeq 263 91 1185 3000 93.1% chr10 + 121052689 121420833 368145 browser details YourSeq 150 67 359 3000 91.9% chr6 + 122544655 122544982 328 browser details YourSeq 142 99 443 3000 94.5% chr1 + 75259172 75259603 432 browser details YourSeq 139 106 310 3000 91.8% chr10 + 40321161 40321563 403 browser details YourSeq 138 99 358 3000 92.7% chr2 + 173091717 173092095 379 browser details YourSeq 136 96 288 3000 93.1% chr8 + 125477712 125478263 552 browser details YourSeq 131 103 321 3000 83.5% chr10 + 111397237 111397788 552 browser details YourSeq 118 84 289 3000 91.6% chr3 - 147296873 147297224 352 browser details YourSeq 114 104 261 3000 93.9% chr18 - 83018397 83018736 340 browser details YourSeq 113 104 359 3000 93.3% chr2 - 31078587 31078988 402 browser details YourSeq 109 104 290 3000 88.1% chr5 - 110895329 110895828 500 browser details YourSeq 109 67 252 3000 86.1% chr15 + 30370741 30370914 174 browser details YourSeq 108 112 299 3000 83.9% chr8 + 123844200 123844345 146 browser details YourSeq 107 104 359 3000 80.8% chr1 + 5142712 5142863 152 browser details YourSeq 106 96 337 3000 84.3% chr8 - 122862448 122862670 223 browser details YourSeq 105 114 360 3000 94.2% chr7 - 130385915 130801746 415832 browser details YourSeq 104 67 310 3000 87.0% chr9 - 98967916 98968179 264 browser details YourSeq 104 104 290 3000 81.0% chr7 - 64499919 64500062 144 browser details YourSeq 101 1041 1179 3000 86.6% chr6 + 120588040 120588350 311

Note: The 3000 bp section upstream of Exon 1 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% chr5 + 134987103 134990102 3000 browser details YourSeq 174 1227 1514 3000 90.6% chr1 - 112731471 113080363 348893 browser details YourSeq 169 1244 1484 3000 95.8% chr7 - 138884126 139241691 357566 browser details YourSeq 163 1237 1484 3000 94.2% chr8 - 45835809 45836161 353 browser details YourSeq 138 1409 2179 3000 90.6% chr1 - 86292725 86483365 190641 browser details YourSeq 133 1318 1484 3000 89.3% chr12 - 11755373 11755530 158 browser details YourSeq 118 1230 1437 3000 91.5% chr2 - 104486947 104487367 421 browser details YourSeq 116 1362 1855 3000 82.9% chr6 - 122679072 122679492 421 browser details YourSeq 115 1749 2185 3000 79.8% chr9 - 65670092 65670457 366 browser details YourSeq 115 1355 1617 3000 83.5% chr15 + 8540566 8540735 170 browser details YourSeq 114 1359 1510 3000 85.9% chr15 + 96786646 96786791 146 browser details YourSeq 110 1240 1372 3000 90.7% chr12 - 72491789 72491913 125 browser details YourSeq 105 1731 2186 3000 78.1% chr1 - 55642899 55643267 369 browser details YourSeq 102 1375 1484 3000 92.5% chr12 + 51450222 51450327 106 browser details YourSeq 97 1227 1377 3000 85.4% chr4 - 51240442 51240574 133 browser details YourSeq 96 1753 2186 3000 77.4% chr1 + 82818280 82818643 364 browser details YourSeq 93 1211 1340 3000 91.7% chr2 + 97526153 97526622 470 browser details YourSeq 92 1227 1355 3000 89.7% chr14 - 88782407 88782533 127 browser details YourSeq 92 1357 1484 3000 92.5% chr6 + 115593563 115593694 132 browser details YourSeq 92 1233 1344 3000 94.3% chr4 + 70513609 70513792 184

Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Cldn3 3 [ Mus musculus (house mouse) ] Gene ID: 12739, updated on 10-Oct-2019

Gene summary

Official Symbol Cldn3 provided by MGI Official Full Name claudin 3 provided by MGI Primary source MGI:MGI:1329044 See related Ensembl:ENSMUSG00000070473 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 mRVP1; Cpetr2; AI182374 Summary This gene encodes a member of the claudin family. are integral membrane and components of tight Orthologs junction strands. Tight junction strands serve as a physical barrier to prevent solutes and water from passing freely through the paracellular space between epithelial or endothelial cell sheets, and also play critical roles in maintaining cell polarity and signal transductions. The protein encoded by this gene is a low-affinity receptor for clostridium perfringens enterotoxin (CPE) produced by the bacterium Clostridium perfringens, and the interaction with CPE results in increased membrane permeability by forming small pores in plasma membrane. This protein is highly overexpressed in uterine carcinosarcoma. This protein is also predominantly present in brain endothelial cells, where it plays a specific role in the establishment and maintenance of blood brain barrier tight junction morphology. [provided by RefSeq, Aug 2012] human all

Genomic context

Location: 5 G2; 5 74.93 cM See Cldn3 in Genome Data Viewer

Exon count: 1

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (134986214..134987476)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (135462084..135463342)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 1 transcript

Gene: Cldn3 ENSMUSG00000070473

Description claudin 3 [Source:MGI Symbol;Acc:MGI:1329044] Gene Synonyms Cpetr2 Location Chromosome 5: 134,986,214-134,987,472 forward strand. GRCm38:CM000998.2 About this gene This gene has 1 transcript (splice variant), 145 orthologues, 40 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cldn3-201 ENSMUST00000094245.3 1259 219aa ENSMUSP00000091799.2 Protein coding CCDS19729 Q545A5 Q9Z0G9 TSL:NA GENCODE basic APPRIS P1

21.26 kb Forward strand

134.980Mb 134.985Mb 134.990Mb 134.995Mb (Comprehensive set... Cldn3-201 >protein coding

Contigs AC084109.2 >

Genes < Wbscr25-202lncRNA (Comprehensive set...

< Wbscr25-201lncRNA

Regulatory Build

134.980Mb 134.985Mb 134.990Mb 134.995Mb Reverse strand 21.26 kb

Regulation Legend

Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000094245

1.26 kb Forward strand

Cldn3-201 >protein coding

ENSMUSP00000091... Transmembrane heli... PDB-ENSP mappings Low complexity (Seg) Prints PR01077

Claudin-3 Pfam PMP-22/EMP/MP20/Claudin superfamily PROSITE patterns Claudin, conserved site PANTHER PTHR12002:SF112

Claudin Gene3D 1.20.140.150

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 219

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

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