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Mouse P2rx7 Knockout Project (CRISPR/Cas9)

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Mouse P2rx7 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse P2rx7 Knockout Project (CRISPR/Cas9) Objective: To create a P2rx7 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The P2rx7 gene (NCBI Reference Sequence: NM_011027 ; Ensembl: ENSMUSG00000029468 ) is located on Mouse chromosome 5. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000100737). 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: Mice homozygous for disruptions in this gene are fertile and viable with no obvious phenotypic abnormality. Cellular responses of macrophages to extracellular ATP are frequently normal however. In addition, long bones are thinner than normal in adult mice. Exon 2 starts from about 7.06% of the coding region. Exon 2~4 covers 17.42% of the coding region. The size of effective KO region: ~4358 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 13 Legends Exon of mouse P2rx7 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 1623 bp section downstream of Exon 4 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. 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(27.05% 541) | C(21.9% 438) | T(28.15% 563) | G(22.9% 458) 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(1623bp) | A(22.86% 371) | C(25.45% 413) | T(30.07% 488) | G(21.63% 351) Note: The 1623 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 + 122650728 122652727 2000 browser details YourSeq 146 231 410 2000 90.2% chr10 - 117732030 117732195 166 browser details YourSeq 145 231 391 2000 92.9% chr4 - 137264747 137264901 155 browser details YourSeq 145 209 410 2000 90.9% chr7 + 143561101 143561301 201 browser details YourSeq 144 231 401 2000 96.2% chr17 + 71484441 71484612 172 browser details YourSeq 142 234 408 2000 90.6% chr6 - 119425763 119425926 164 browser details YourSeq 141 228 410 2000 87.9% chr4 + 41342265 41342435 171 browser details YourSeq 141 234 408 2000 89.4% chr13 + 61773600 61773760 161 browser details YourSeq 140 234 408 2000 90.0% chr13 - 62569030 62569197 168 browser details YourSeq 138 231 405 2000 87.7% chr10 - 26573538 26573702 165 browser details YourSeq 137 252 410 2000 91.9% chr10 - 76896511 76896662 152 browser details YourSeq 137 231 411 2000 90.8% chr10 + 45267013 45267191 179 browser details YourSeq 136 231 392 2000 90.4% chr12 - 102517259 102517415 157 browser details YourSeq 136 234 397 2000 90.5% chr17 + 79099004 79099163 160 browser details YourSeq 135 233 392 2000 92.0% chr8 - 22412900 22413054 155 browser details YourSeq 135 234 392 2000 91.5% chr19 - 43852793 43852946 154 browser details YourSeq 135 233 405 2000 94.2% chr13 - 61999606 62118759 119154 browser details YourSeq 134 234 392 2000 89.6% chr1 + 152798074 152798226 153 browser details YourSeq 133 233 410 2000 92.8% chr5 - 138767501 138767707 207 browser details YourSeq 133 231 392 2000 89.6% chr5 + 21685491 21685635 145 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 1623 1 1623 1623 100.0% chr5 + 122657086 122658708 1623 browser details YourSeq 281 843 1415 1623 89.3% chr1 + 181924190 181924559 370 browser details YourSeq 275 866 1423 1623 86.5% chr17 - 32088419 32088762 344 browser details YourSeq 268 845 1416 1623 88.7% chr2 - 150129712 150130184 473 browser details YourSeq 263 840 1423 1623 92.1% chr16 + 11165486 11166196 711 browser details YourSeq 259 839 1415 1623 86.5% chr8 - 70101281 70101625 345 browser details YourSeq 249 847 1412 1623 92.6% chr9 - 89086869 89087456 588 browser details YourSeq 244 845 1414 1623 92.7% chrX + 104045751 104046378 628 browser details YourSeq 243 854 1411 1623 88.1% chr7 + 141045847 141046353 507 browser details YourSeq 225 855 1412 1623 87.5% chr5 + 110622434 110622840 407 browser details YourSeq 221 843 1415 1623 88.2% chr11 - 33069511 33070140 630 browser details YourSeq 219 849 1366 1623 90.8% chr4 - 115947320 115947876 557 browser details YourSeq 211 858 1388 1623 84.9% chr11 - 101609282 101609631 350 browser details YourSeq 211 899 1412 1623 83.3% chr1 + 133036154 133036445 292 browser details YourSeq 206 843 1350 1623 84.7% chr13 - 93565323 93565653 331 browser details YourSeq 203 844 1319 1623 92.1% chr11 - 51758276 51758948 673 browser details YourSeq 200 843 1413 1623 86.0% chr6 - 39043805 39044162 358 browser details YourSeq 198 838 1350 1623 84.1% chr9 - 70556950 70557395 446 browser details YourSeq 193 848 1361 1623 87.8% chr14 - 98393577 98394131 555 browser details YourSeq 191 849 1415 1623 90.7% chr11 + 23321407 23322053 647 Note: The 1623 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: P2rx7 purinergic receptor P2X, ligand-gated ion channel, 7 [ Mus musculus (house mouse) ] Gene ID: 18439, updated on 24-Oct-2019 Gene summary Official Symbol P2rx7 provided by MGI Official Full Name purinergic receptor P2X, ligand-gated ion channel, 7 provided by MGI Primary source MGI:MGI:1339957 See related Ensembl:ENSMUSG00000029468 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 P2X7R; P2X(7); AI467586 Expression Ubiquitous expression in subcutaneous fat pad adult (RPKM 4.7), lung adult (RPKM 4.4) and 28 other tissues See more Orthologs human all Genomic context Location: 5; 5 F See P2rx7 in Genome Data Viewer Exon count: 16 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (122643927..122692336) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (123093920..123141441) Chromosome 5 - NC_000071.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: P2rx7 ENSMUSG00000029468 Description purinergic receptor P2X, ligand-gated ion channel, 7 [Source:MGI Symbol;Acc:MGI:1339957] Gene Synonyms P2X(7), P2X7 receptor, P2X7R Location Chromosome 5: 122,643,911-122,691,432 forward strand. GRCm38:CM000998.2 About this gene This gene has 8 transcripts (splice variants), 174 orthologues, 6 paralogues, is a member of 1 Ensembl protein family and is associated with 19 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags P2rx7- ENSMUST00000100737.9 4936 595aa ENSMUSP00000098303.3 Protein coding CCDS19652 Q8CHP4 TSL:1 203 Q9Z1M0 GENCODE basic APPRIS P1 P2rx7- ENSMUST00000121489.7 2576 431aa ENSMUSP00000112440.1 Protein coding CCDS51644 Q3UN00 TSL:1 204 GENCODE basic P2rx7- ENSMUST00000031425.14 1605 442aa ENSMUSP00000031425.8 Protein coding CCDS19651 F8WI93 TSL:1 201 GENCODE basic P2rx7- ENSMUST00000086247.5 712 153aa ENSMUSP00000083425.5 Protein coding CCDS19653 Q6P5P0 TSL:1 202 GENCODE basic P2rx7- ENSMUST00000199371.1 1848 592aa ENSMUSP00000158311.1 Protein coding - C8YIX4 TSL:1 208 GENCODE basic P2rx7- ENSMUST00000197042.1 2103 No - Retained - - TSL:NA 205 protein intron P2rx7- ENSMUST00000199281.1 480 No - lncRNA - - TSL:5 207 protein P2rx7- ENSMUST00000197102.1 433 No - lncRNA - - TSL:3 206 protein Page 7 of 9 https://www.alphaknockout.com 67.52 kb Forward strand 122.64Mb 122.66Mb 122.68Mb 122.70Mb Genes (Comprehensive set... P2rx7-204 >protein coding P2rx7-203 >protein coding P2rx7-201 >protein coding P2rx7-202 >protein coding P2rx7-206 >lncRNA P2rx7-207 >lncRNA P2rx7-205 >retained intron P2rx7-208 >protein coding Mir8115-201 >miRNA Contigs AC114632.9 > Genes < Gm10064-201processed pseudogene (Comprehensive set... Regulatory Build 122.64Mb 122.66Mb 122.68Mb 122.70Mb Reverse strand 67.52 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 pseudogene RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000100737 40.38 kb Forward strand P2rx7-203 >protein coding ENSMUSP00000098... Low complexity (Seg) TIGRFAM P2X purinoreceptor Prints P2X7 purinoceptor P2X purinoreceptor Pfam PF00864 PROSITE patterns P2X purinoreceptor PANTHER PTHR10125 P2X7 purinoceptor Gene3D 1.10.287.940 P2X purinoreceptor extracellular domain superfamily All sequence SNPs/i..
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