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Mouse Pcnx Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Pcnx gene (NCBI Reference Sequence: NM_018814 ; Ensembl: ENSMUSG00000021140 ) is located on Mouse chromosome 12. 36 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 36 (Transcript: ENSMUST00000021567). 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 Pcnx gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-66I1 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 2.19% 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: 34458 bp, and the size of intron 2 for 3'-loxP site insertion: 11257 bp. The size of effective cKO region: ~709 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 36 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pcnx 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(7209bp) | A(26.0% 1874) | C(20.82% 1501) | T(32.63% 2352) | G(20.56% 1482)

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% chr12 + 81891734 81894733 3000 browser details YourSeq 391 2087 2548 3000 97.6% chr4 + 49008315 49009041 727 browser details YourSeq 390 1968 2545 3000 93.7% chr5 - 22821147 22821593 447 browser details YourSeq 389 2140 2548 3000 97.1% chr7 + 108132974 108133381 408 browser details YourSeq 389 2140 2548 3000 97.1% chr7 + 41049678 41050084 407 browser details YourSeq 389 2140 2548 3000 97.8% chr17 + 68687337 68687816 480 browser details YourSeq 388 2142 2548 3000 97.3% chrX - 87574502 87574907 406 browser details YourSeq 388 2140 2548 3000 97.6% chr14 - 52813265 52813696 432 browser details YourSeq 387 2140 2548 3000 96.9% chr6 + 60113443 60113850 408 browser details YourSeq 385 2140 2548 3000 96.4% chr1 - 86780730 86781135 406 browser details YourSeq 384 2139 2548 3000 95.9% chr19 - 35469773 35470179 407 browser details YourSeq 383 2144 2548 3000 97.6% chr9 - 96373055 96373474 420 browser details YourSeq 383 2145 2548 3000 97.8% chr11 - 13188737 13189145 409 browser details YourSeq 383 2144 2548 3000 97.8% chr1 + 107058357 107058762 406 browser details YourSeq 382 2148 2548 3000 97.8% chr16 + 12621850 12622250 401 browser details YourSeq 382 2149 2548 3000 97.8% chr11 + 80187254 80187653 400 browser details YourSeq 382 2144 2548 3000 97.3% chr1 + 56521067 56521473 407 browser details YourSeq 381 2147 2548 3000 97.6% chr9 - 79279299 79279720 422 browser details YourSeq 381 2134 2548 3000 95.8% chrX + 18648872 18649277 406 browser details YourSeq 381 2152 2548 3000 98.0% chr10 + 111513041 111513437 397

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% chr12 + 81895443 81898442 3000 browser details YourSeq 112 1029 1752 3000 86.8% chr16 + 6040693 6403705 363013 browser details YourSeq 88 1147 1727 3000 86.2% chr9 - 114521094 114835213 314120 browser details YourSeq 83 1143 1260 3000 85.6% chr14 - 70506789 70506907 119 browser details YourSeq 83 1152 1264 3000 88.2% chr11 - 78964173 78964293 121 browser details YourSeq 81 1126 1245 3000 86.0% chr3 - 120888104 120888227 124 browser details YourSeq 78 1143 1250 3000 86.2% chr11 + 6312215 6312322 108 browser details YourSeq 77 1141 1725 3000 76.2% chr14 - 30401518 30401840 323 browser details YourSeq 76 1143 1250 3000 82.7% chr8 - 3463604 3463708 105 browser details YourSeq 75 1156 1250 3000 92.3% chr3 + 138591376 138591470 95 browser details YourSeq 74 1143 1260 3000 83.5% chr7 - 126451775 126451891 117 browser details YourSeq 74 1147 1247 3000 87.2% chr5 - 145111122 145111223 102 browser details YourSeq 72 1152 1250 3000 86.9% chr13 + 38120234 38120333 100 browser details YourSeq 71 1147 1260 3000 82.2% chr4 - 116862871 116862987 117 browser details YourSeq 71 1152 1247 3000 87.5% chr2 - 5994786 5994882 97 browser details YourSeq 71 1146 1249 3000 87.7% chr1 + 134951413 134951517 105 browser details YourSeq 70 1143 1250 3000 82.5% chr18 - 34452601 34452708 108 browser details YourSeq 70 1129 1252 3000 86.4% chr3 + 52012315 52012441 127 browser details YourSeq 69 1143 1250 3000 85.6% chr2 - 132676643 132676751 109 browser details YourSeq 69 1143 1249 3000 85.8% chr1 - 59243598 59243704 107

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: Pcnx pecanex homolog [ Mus musculus (house mouse) ] Gene ID: 54604, updated on 12-Aug-2019

Gene summary

Official Symbol Pcnx provided by MGI Official Full Name pecanex homolog provided by MGI Primary source MGI:MGI:1891924 See related Ensembl:ENSMUSG00000021140 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 Pcnx1; Pcnxl1; AF096286; AI327143; AI413187; 2900024E21Rik; 3526401J03Rik Expression Ubiquitous expression in lung adult (RPKM 13.9), ovary adult (RPKM 11.9) and 26 other tissues See more Orthologs human all

Genomic context

Location: 12; 12 D1 See Pcnx in Genome Data Viewer

Exon count: 36

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (81859913..82000924)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (82961017..83101911)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 8 transcripts

Gene: Pcnx ENSMUSG00000021140

Description pecanex homolog [Source:MGI Symbol;Acc:MGI:1891924] Gene Synonyms 2900024E21Rik, 3526401J03Rik Location Chromosome 12: 81,860,023-82,000,924 forward strand. GRCm38:CM001005.2 About this gene This gene has 8 transcripts (splice variants), 202 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pcnx- ENSMUST00000021567.5 12139 2344aa ENSMUSP00000021567.5 Protein coding CCDS26025 Q9QYC1 TSL:2 201 GENCODE basic APPRIS P2

Pcnx- ENSMUST00000221721.1 8318 2338aa ENSMUSP00000152104.1 Protein coding - A0A1Y7VKR5 TSL:5 204 GENCODE basic APPRIS ALT2

Pcnx- ENSMUST00000221675.1 4812 1473aa ENSMUSP00000152385.1 Protein coding - A0A1Y7VLV8 CDS 5' 203 incomplete TSL:1

Pcnx- ENSMUST00000222005.1 7827 818aa ENSMUSP00000152302.1 Nonsense mediated - A0A1Y7VJ77 TSL:5 205 decay

Pcnx- ENSMUST00000222468.1 685 75aa ENSMUSP00000152168.1 Nonsense mediated - A0A1Y7VLG1 CDS 5' 206 decay incomplete TSL:3

Pcnx- ENSMUST00000222828.1 3147 No - Retained intron - - TSL:NA 207 protein

Pcnx- ENSMUST00000221472.1 370 No - Retained intron - - TSL:2 202 protein

Pcnx- ENSMUST00000222908.1 347 No - Retained intron - - TSL:3 208 protein

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160.90 kb Forward strand 81.90Mb 81.95Mb 82.00Mb Genes (Comprehensive set... Gm47080-201 >lncRNA Pcnx-203 >protein coding

Pcnx-204 >protein coding

Pcnx-201 >protein coding

Pcnx-205 >nonsense mediated decay

Pcnx-207 >retained intron Pcnx-202 >retained intron

Pcnx-208 >retained intron

Pcnx-206 >nonsense mediated decay

Gm49749-201 >lncRNA

Contigs < AC124595.4 < AC124484.5 Regulatory Build

81.90Mb 81.95Mb 82.00Mb Reverse strand 160.90 kb

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

Gene Legend Protein Coding

Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000021567

140.90 kb Forward strand

Pcnx-201 >protein coding

ENSMUSP00000021... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam Pecanex, C-terminal

PANTHER Protein Pecanex

PTHR12372:SF2

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

Variant Legend

frameshift variant missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2344

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

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