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Mouse P2rx4 Knockout Project (CRISPR/Cas9)

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Mouse P2rx4 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse P2rx4 Knockout Project (CRISPR/Cas9) Objective: To create a P2rx4 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The P2rx4 gene (NCBI Reference Sequence: NM_011026 ; Ensembl: ENSMUSG00000029470 ) is located on Mouse chromosome 5. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000031429). Exon 2~4 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Homozygous mutation of this gene results in hypertension, abnormal artery morphology, abnormal nitric oxide homeostasis, and impaired flow induced vascular remodeling and vasodilation. Exon 2 starts from about 11.6% of the coding region. Exon 2~4 covers 25.17% of the coding region. The size of effective KO region: ~4175 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 12 Legends Exon of mouse P2rx4 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 556 bp section downstream of Exon 4 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(27.95% 559) | C(22.7% 454) | T(27.05% 541) | G(22.3% 446) Note: The 2000 bp section upstream of Exon 2 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(556bp) | A(23.02% 128) | C(29.14% 162) | T(23.74% 132) | G(24.1% 134) Note: The 556 bp section downstream of Exon 4 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. Page 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr5 + 122712400 122714399 2000 browser details YourSeq 582 795 1557 2000 91.0% chr15 - 37064304 37065301 998 browser details YourSeq 582 793 1541 2000 92.4% chr1 - 44971148 44971959 812 browser details YourSeq 577 796 1531 2000 92.4% chr1 - 23936773 23937762 990 browser details YourSeq 519 343 1351 2000 91.8% chr11 - 52357134 52357803 670 browser details YourSeq 519 793 1458 2000 93.7% chr19 + 3747449 3748222 774 browser details YourSeq 509 795 1499 2000 92.3% chr16 + 23097854 23098609 756 browser details YourSeq 506 796 1405 2000 93.8% chr13 - 114593712 114594434 723 browser details YourSeq 506 298 1339 2000 90.4% chr12 - 114464469 114465422 954 browser details YourSeq 502 796 1358 2000 94.7% chr13 + 21900950 21901514 565 browser details YourSeq 501 298 1338 2000 89.5% chr16 + 4797907 4798614 708 browser details YourSeq 500 794 1351 2000 94.9% chr14 - 10168973 10169530 558 browser details YourSeq 496 796 1351 2000 94.7% chr1 - 156404923 156405478 556 browser details YourSeq 495 794 1351 2000 94.5% chr3 - 55106639 55107197 559 browser details YourSeq 494 796 1351 2000 94.5% chr14 - 76241735 76242290 556 browser details YourSeq 490 796 1349 2000 94.3% chr17 + 34310285 34310838 554 browser details YourSeq 489 794 1351 2000 94.1% chr17 - 72479216 72479777 562 browser details YourSeq 489 778 1536 2000 89.3% chr16 - 47700299 47701111 813 browser details YourSeq 489 795 1351 2000 94.9% chr7 + 86873384 86873955 572 browser details YourSeq 488 796 1353 2000 93.7% chr5 + 20472749 20473304 556 Note: The 2000 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 556 1 556 556 100.0% chr5 + 122718575 122719130 556 browser details YourSeq 25 163 187 556 100.0% chr16 - 46486992 46487016 25 browser details YourSeq 25 165 192 556 96.5% chr1 - 87843395 87843437 43 browser details YourSeq 25 163 193 556 90.4% chr1 - 39714641 39714671 31 browser details YourSeq 25 158 184 556 96.3% chr7 + 27423021 27423047 27 browser details YourSeq 24 158 181 556 100.0% chrX + 73720051 73720074 24 browser details YourSeq 24 159 182 556 100.0% chr17 + 67755609 67755632 24 browser details YourSeq 24 166 229 556 68.8% chr10 + 67131266 67131329 64 browser details YourSeq 23 160 182 556 100.0% chrX - 155296924 155296946 23 browser details YourSeq 23 160 182 556 100.0% chr6 - 29460369 29460391 23 browser details YourSeq 23 159 181 556 100.0% chr13 - 75824659 75824681 23 browser details YourSeq 23 159 181 556 100.0% chr6 + 40242380 40242402 23 browser details YourSeq 23 160 182 556 100.0% chr1 + 63380476 63380498 23 browser details YourSeq 22 159 180 556 100.0% chrX - 36897125 36897146 22 browser details YourSeq 22 160 181 556 100.0% chr8 - 25828576 25828597 22 browser details YourSeq 22 160 181 556 100.0% chr2 - 35308696 35308717 22 browser details YourSeq 22 160 181 556 100.0% chr12 + 84977176 84977197 22 browser details YourSeq 22 160 181 556 100.0% chr11 + 49667006 49667027 22 browser details YourSeq 22 380 402 556 100.0% chr10 + 123472405 123472434 30 browser details YourSeq 22 158 180 556 100.0% chr1 + 191215507 191215531 25 Note: The 556 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: P2rx4 purinergic receptor P2X, ligand-gated ion channel 4 [ Mus musculus (house mouse) ] Gene ID: 18438, updated on 10-Oct-2019 Gene summary Official Symbol P2rx4 provided by MGI Official Full Name purinergic receptor P2X, ligand-gated ion channel 4 provided by MGI Primary source MGI:MGI:1338859 See related Ensembl:ENSMUSG00000029470 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 P2X4; AI504491; AW555605; D5Ertd444e Expression Ubiquitous expression in colon adult (RPKM 57.8), lung adult (RPKM 28.0) and 23 other tissues See more Orthologs human all Genomic context Location: 5 F; 5 62.53 cM See P2rx4 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (122707518..122729739) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (123157566..123179053) Chromosome 5 - NC_000071.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: P2rx4 ENSMUSG00000029470 Description purinergic receptor P2X, ligand-gated ion channel 4 [Source:MGI Symbol;Acc:MGI:1338859] Gene Synonyms D5Ertd444e, P2X4 Location Chromosome 5: 122,707,544-122,729,738 forward strand. GRCm38:CM000998.2 About this gene This gene has 9 transcripts (splice variants), 202 orthologues, 6 paralogues, is a member of 1 Ensembl protein family and is associated with 13 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags P2rx4- ENSMUST00000031429.13 2702 388aa ENSMUSP00000031429.7 Protein coding CCDS19654 Q9Z257 TSL:1 201 GENCODE basic APPRIS P1 P2rx4- ENSMUST00000081554.12 1887 361aa ENSMUSP00000080269.6 Protein coding CCDS80393 Q9Z256 TSL:1 202 GENCODE basic P2rx4- ENSMUST00000142664.2 1111 366aa ENSMUSP00000117193.2 Protein coding CCDS80392 D3Z5U5 TSL:1 206 GENCODE basic P2rx4- ENSMUST00000139631.7 1030 339aa ENSMUSP00000118163.2 Protein coding - D3YYR5 TSL:5 204 GENCODE basic P2rx4- ENSMUST00000198560.1 810 52aa ENSMUSP00000142849.1 Nonsense mediated - A0A0G2JEP2 TSL:5 209 decay P2rx4- ENSMUST00000195963.1 3895 No - Retained intron - - TSL:NA 208 protein P2rx4- ENSMUST00000152337.5 711 No - Retained intron - - TSL:3 207 protein P2rx4- ENSMUST00000132062.1 513 No - Retained intron - - TSL:3 203 protein P2rx4- ENSMUST00000139712.1 476 No - lncRNA - - TSL:2 205 protein Page 7 of 9 https://www.alphaknockout.com 42.20 kb Forward strand 122.70Mb 122.71Mb 122.72Mb 122.73Mb Genes (Comprehensive set... P2rx4-201 >protein coding P2rx4-208 >retained intron P2rx4-203 >retained intron P2rx4-207 >retained intron P2rx4-205 >lncRNA P2rx4-209 >nonsense mediated decay P2rx4-202 >protein coding P2rx4-206 >protein coding P2rx4-204 >protein coding Contigs < AC115728.11 Genes < Gm10064-201processed pseudogene < Camkk2-201protein coding (Comprehensive set... < Camkk2-211protein coding < Camkk2-204protein coding < Camkk2-203protein coding Regulatory Build 122.70Mb 122.71Mb 122.72Mb 122.73Mb Reverse strand 42.20 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000031429 22.20 kb Forward strand P2rx4-201 >protein coding ENSMUSP00000031... Transmembrane heli... Low complexity (Seg) TIGRFAM P2X purinoreceptor Prints P2X purinoreceptor P2X4 purinoceptor Pfam PF00864 PROSITE patterns P2X purinoreceptor PIRSF P2X purinoreceptor PANTHER PTHR10125 PTHR10125:SF18 Gene3D P2X purinoreceptor extracellular domain superfamily 1.10.287.940 All sequence SNPs/i..
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