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Mouse Plxdc1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Plxdc1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Plxdc1 gene (NCBI Reference Sequence: NM_001163608 ; Ensembl: ENSMUSG00000017417 ) 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: ENSMUST00000107565). 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 Plxdc1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-318C24 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 2 starts from about 5.26% 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: 7556 bp, and the size of intron 2 for 3'-loxP site insertion: 21915 bp. The size of effective cKO region: ~754 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 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Plxdc1 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(7179bp) | A(24.32% 1746) | C(23.46% 1684) | T(24.01% 1724) | G(28.21% 2025)

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 - 97978932 97981931 3000 browser details YourSeq 111 40 2257 3000 90.6% chr11 - 68542901 68823164 280264 browser details YourSeq 104 20 2257 3000 87.7% chr10 - 128420001 128623251 203251 browser details YourSeq 88 49 2208 3000 89.3% chr11 - 5174020 5532473 358454 browser details YourSeq 68 20 109 3000 87.8% chr1 - 191017059 191017148 90 browser details YourSeq 65 20 102 3000 89.7% chr9 - 110714896 110714977 82 browser details YourSeq 64 40 117 3000 91.1% chr7 - 79491594 79491671 78 browser details YourSeq 63 18 102 3000 81.6% chr6 + 146638987 146639062 76 browser details YourSeq 63 7 102 3000 88.4% chr1 + 60412985 60413079 95 browser details YourSeq 62 20 101 3000 87.9% chr7 - 28643846 28643927 82 browser details YourSeq 62 17 102 3000 86.1% chr1 - 135506806 135506891 86 browser details YourSeq 62 20 101 3000 87.9% chr7 + 24627994 24628075 82 browser details YourSeq 62 18 102 3000 81.1% chr11 + 98957666 98957739 74 browser details YourSeq 61 14 102 3000 84.3% chr12 + 72878060 72878148 89 browser details YourSeq 61 18 102 3000 80.3% chr11 + 91050863 91050938 76 browser details YourSeq 60 34 109 3000 89.5% chrX - 139633714 139633789 76 browser details YourSeq 60 34 109 3000 85.2% chr7 - 101814755 101814828 74 browser details YourSeq 60 17 95 3000 95.6% chr16 - 96203070 96203274 205 browser details YourSeq 59 20 102 3000 87.4% chr4 - 154615371 154615451 81 browser details YourSeq 59 20 93 3000 90.6% chr7 + 144891236 144891310 75

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 - 97975253 97978252 3000 browser details YourSeq 524 2233 3000 3000 88.8% chr4 - 153740888 153741589 702 browser details YourSeq 515 2232 3000 3000 86.5% chr12 + 40172086 40172806 721 browser details YourSeq 500 2233 3000 3000 85.3% chr7 + 110557754 110558462 709 browser details YourSeq 490 2236 3000 3000 87.1% chr7 - 142766068 142766783 716 browser details YourSeq 469 2233 2998 3000 87.0% chr7 + 46191807 46192512 706 browser details YourSeq 469 2234 2998 3000 85.9% chr15 + 59289085 59289725 641 browser details YourSeq 461 2265 3000 3000 88.1% chr13 + 80812263 80813015 753 browser details YourSeq 460 2240 2993 3000 87.7% chr3 - 101787174 101787857 684 browser details YourSeq 457 2230 3000 3000 84.5% chr4 - 137417003 137417697 695 browser details YourSeq 456 2259 3000 3000 84.2% chr2 + 6254257 6254935 679 browser details YourSeq 454 2233 2997 3000 86.9% chr1 + 183522686 183523380 695 browser details YourSeq 453 2232 3000 3000 85.2% chr17 + 73368854 73369574 721 browser details YourSeq 451 2281 2988 3000 87.3% chr13 - 56318750 56319442 693 browser details YourSeq 445 2238 2998 3000 88.0% chr16 + 23158371 23159094 724 browser details YourSeq 442 2238 3000 3000 85.3% chr2 + 16517627 16518367 741 browser details YourSeq 441 2234 2976 3000 88.8% chr4 - 85393637 85394378 742 browser details YourSeq 439 2238 3000 3000 84.2% chr17 + 17714168 17714884 717 browser details YourSeq 437 2249 2987 3000 85.0% chr6 - 123737350 123738047 698 browser details YourSeq 437 2233 3000 3000 87.7% chr13 - 6294713 6295475 763

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: Plxdc1 plexin domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 72324, updated on 12-Aug-2019

Gene summary

Official Symbol Plxdc1 provided by MGI Official Full Name plexin domain containing 1 provided by MGI Primary source MGI:MGI:1919574 See related Ensembl:ENSMUSG00000017417 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 Tem7; AI848450; 2410003I07Rik Expression Biased expression in testis adult (RPKM 22.2), thymus adult (RPKM 19.3) and 12 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 D See Plxdc1 in Genome Data Viewer

Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (97923237..97986669, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (97784551..97847760, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 4 transcripts

Gene: Plxdc1 ENSMUSG00000017417

Description plexin domain containing 1 [Source:MGI Symbol;Acc:MGI:1919574] Gene Synonyms 2410003I07Rik, Tem7 Location Chromosome 11: 97,923,238-97,986,444 reverse strand. GRCm38:CM001004.2 About this gene This gene has 4 transcripts (splice variants), 243 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Plxdc1-201 ENSMUST00000017561.14 2881 500aa ENSMUSP00000017561.8 Protein coding CCDS25333 Q91ZV7 TSL:1 GENCODE basic APPRIS P1

Plxdc1-203 ENSMUST00000107565.2 2439 507aa ENSMUSP00000103191.2 Protein coding CCDS48897 Q91ZV7 TSL:1 GENCODE basic

Plxdc1-202 ENSMUST00000107564.1 619 111aa ENSMUSP00000103190.1 Protein coding - A2A539 TSL:2 GENCODE basic

Plxdc1-204 ENSMUST00000141708.1 2685 No protein - lncRNA - - TSL:1

83.21 kb Forward strand

Genes Gm11633-201 >processed pseudogene (Comprehensive set...

Contigs AL591209.17 > Genes (Comprehensive set... < Plxdc1-201protein coding

< Plxdc1-203protein coding

< Plxdc1-204lncRNA

< Gm22461-201miRNA < Plxdc1-202protein coding

< Arl5c-201protein coding

< Arl5c-202protein coding

Regulatory Build

Reverse strand 83.21 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene pseudogene

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Transcript: ENSMUST00000107565

< Plxdc1-203protein coding

Reverse strand 62.74 kb

ENSMUSP00000103... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Pfam Plexin repeat PANTHER Plexin domain-containing protein

Plexin domain-containing protein 1

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

Variant Legend splice acceptor variant missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 507

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

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