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Mouse Pde9a Knockout Project (CRISPR/Cas9)

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Mouse Pde9a Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pde9a Knockout Project (CRISPR/Cas9) Objective: To create a Pde9a knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pde9a gene (NCBI Reference Sequence: NM_008804 ; Ensembl: ENSMUSG00000041119 ) is located on Mouse chromosome 17. 19 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 18 (Transcript: ENSMUST00000047168). Exon 8~13 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 a null allele exhibit suppressed pressure-overload-induced cardiac pathobiology. Exon 8 starts from about 29.59% of the coding region. Exon 8~13 covers 36.58% of the coding region. The size of effective KO region: ~8326 bp. The KO region does not have any other known gene. Page 1 of 10 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 8 9 10 11 12 13 19 Legends Exon of mouse Pde9a Knockout region Page 2 of 10 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 8 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 13 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 10 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(25.9% 518) | C(20.95% 419) | T(27.1% 542) | G(26.05% 521) Note: The 2000 bp section upstream of Exon 8 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(2000bp) | A(24.9% 498) | C(26.65% 533) | T(23.25% 465) | G(25.2% 504) Note: The 2000 bp section downstream of Exon 13 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 10 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% chr17 + 31451403 31453402 2000 browser details YourSeq 250 835 1461 2000 88.4% chr4 + 116969204 116978941 9738 browser details YourSeq 239 833 1460 2000 93.6% chr12 + 111366346 111642511 276166 browser details YourSeq 220 835 1461 2000 82.7% chr2 + 140158140 140158465 326 browser details YourSeq 220 828 1461 2000 85.3% chr2 + 118610340 118610669 330 browser details YourSeq 220 843 1460 2000 83.8% chr1 + 74673640 74673957 318 browser details YourSeq 218 835 1460 2000 82.7% chr16 - 13956815 13957162 348 browser details YourSeq 218 835 1460 2000 81.9% chr2 + 71009795 71010175 381 browser details YourSeq 212 835 1461 2000 83.8% chr1 + 13230083 13230367 285 browser details YourSeq 198 835 1409 2000 86.6% chr1 - 36671777 36672294 518 browser details YourSeq 194 843 1397 2000 81.9% chr4 + 126941377 126941822 446 browser details YourSeq 189 835 1440 2000 83.6% chr15 - 99835900 99836397 498 browser details YourSeq 187 835 1457 2000 89.8% chr11 - 69431384 69432003 620 browser details YourSeq 178 835 1409 2000 84.3% chr2 - 174541910 174542197 288 browser details YourSeq 175 834 1378 2000 83.5% chr4 + 136091019 136091256 238 browser details YourSeq 173 835 1354 2000 91.4% chr17 + 36993294 36993879 586 browser details YourSeq 172 542 1014 2000 85.1% chr7 + 92378202 92378481 280 browser details YourSeq 171 812 1023 2000 91.4% chr11 - 61625968 61626386 419 browser details YourSeq 171 835 1464 2000 83.6% chr8 + 72450791 72450998 208 browser details YourSeq 170 843 1199 2000 86.9% chr19 - 32734702 32735024 323 Note: The 2000 bp section upstream of Exon 8 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 2000 1 2000 2000 100.0% chr17 + 31461729 31463728 2000 browser details YourSeq 280 371 962 2000 91.4% chr4 - 153122646 153123290 645 browser details YourSeq 263 632 964 2000 88.5% chr11 + 46307485 46307808 324 browser details YourSeq 262 632 962 2000 88.9% chr4 - 150993552 150993877 326 browser details YourSeq 260 632 963 2000 89.1% chr17 + 13452239 13452567 329 browser details YourSeq 257 632 961 2000 88.7% chr2 - 168435081 168435405 325 browser details YourSeq 257 631 962 2000 89.0% chr11 + 103426749 103427074 326 browser details YourSeq 257 635 959 2000 89.5% chr11 + 100534360 100534680 321 browser details YourSeq 256 620 962 2000 89.5% chr19 + 30088612 30089067 456 browser details YourSeq 255 635 967 2000 88.4% chr16 - 14213755 14214069 315 browser details YourSeq 255 631 963 2000 87.7% chr1 + 131028342 131028670 329 browser details YourSeq 254 631 963 2000 88.1% chr2 - 73357995 73358322 328 browser details YourSeq 254 632 962 2000 88.0% chr10 - 80046271 80046593 323 browser details YourSeq 254 631 962 2000 89.1% chr8 + 125284129 125284444 316 browser details YourSeq 253 633 962 2000 89.1% chr6 - 88155785 88156109 325 browser details YourSeq 252 632 963 2000 87.1% chr7 - 111418848 111419174 327 browser details YourSeq 252 632 965 2000 87.4% chr4 - 142096696 142097024 329 browser details YourSeq 252 634 961 2000 88.2% chr18 - 75608255 75608576 322 browser details YourSeq 251 635 963 2000 88.5% chr17 - 8598970 8599294 325 browser details YourSeq 251 632 937 2000 90.7% chr14 - 65146664 65146965 302 Note: The 2000 bp section downstream of Exon 13 is BLAT searched against the genome. No significant similarity is found. Page 5 of 10 https://www.alphaknockout.com Gene and protein information: Pde9a phosphodiesterase 9A [ Mus musculus (house mouse) ] Gene ID: 18585, updated on 12-Aug-2019 Gene summary Official Symbol Pde9a provided by MGI Official Full Name phosphodiesterase 9A provided by MGI Primary source MGI:MGI:1277179 See related Ensembl:ENSMUSG00000041119 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 PDE9A1 Expression Broad expression in colon adult (RPKM 62.7), large intestine adult (RPKM 61.1) and 19 other tissues See more Orthologs human all Genomic context Location: 17; 17 B1 See Pde9a in Genome Data Viewer Exon count: 20 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (31386105..31476309) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (31523179..31613254) Chromosome 17 - NC_000083.6 Page 6 of 10 https://www.alphaknockout.com Transcript information: This gene has 28 transcripts Gene: Pde9a ENSMUSG00000041119 Description phosphodiesterase 9A [Source:MGI Symbol;Acc:MGI:1277179] Gene Synonyms PDE9A1 Location Chromosome 17: 31,386,210-31,476,310 forward strand. GRCm38:CM001010.2 About this gene This gene has 28 transcripts (splice variants), 197 orthologues, 21 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pde9a- ENSMUST00000047168.12 2089 534aa ENSMUSP00000038005.5 Protein coding CCDS28605 O70628 TSL:1 201 GENCODE basic APPRIS P2 Pde9a- ENSMUST00000127929.7 1891 534aa ENSMUSP00000117611.1 Protein coding CCDS28605 O70628 TSL:1 203 GENCODE basic APPRIS P2 Pde9a- ENSMUST00000235639.1 1913 495aa ENSMUSP00000158058.1 Protein coding - A0A494BAJ0 CDS 5' 215 incomplete Pde9a- ENSMUST00000136384.8 1887 387aa ENSMUSP00000116724.2 Protein coding - D3Z408 TSL:2 207 GENCODE basic Pde9a- ENSMUST00000134525.8 1810 508aa ENSMUSP00000121003.1 Protein coding - D3YTQ4 TSL:5 206 GENCODE basic APPRIS ALT2 Pde9a- ENSMUST00000235719.1 1787 452aa ENSMUSP00000158101.1 Protein coding - A0A494BAI4 GENCODE 217 basic Pde9a- ENSMUST00000237363.1 1660 383aa ENSMUSP00000158458.1 Protein coding - A0A494BBD2 GENCODE 224 basic Pde9a- ENSMUST00000236454.1 1640 387aa ENSMUSP00000157420.1 Protein coding - D3Z408 GENCODE 220 basic Pde9a- ENSMUST00000238091.1 1555 377aa ENSMUSP00000157753.1 Protein coding - A0A494B9U5 GENCODE 228 basic Pde9a- ENSMUST00000154392.1 778 180aa ENSMUSP00000117065.1 Protein coding - F6ZAU1 CDS 5' 211 incomplete TSL:3 Pde9a- ENSMUST00000238006.1 591 115aa ENSMUSP00000158100.1 Protein coding - A0A494BAI3 CDS 5' 227 incomplete Pde9a- ENSMUST00000124902.8 1904 102aa ENSMUSP00000118869.1 Nonsense mediated - D6RH57 TSL:5 202 decay Pde9a- ENSMUST00000236850.1 1817 63aa ENSMUSP00000158049.1 Nonsense mediated - D6RGP8 - 222 decay Pde9a- ENSMUST00000131417.8 1762 63aa ENSMUSP00000115188.1 Nonsense mediated - D6RGP8 TSL:5 205 decay Pde9a- ENSMUST00000236077.1 1755 68aa ENSMUSP00000158208.1
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