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Mouse Ppip5k2 Knockout Project (CRISPR/Cas9)

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Mouse Ppip5k2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ppip5k2 Knockout Project (CRISPR/Cas9) Objective: To create a Ppip5k2 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ppip5k2 gene (NCBI Reference Sequence: NM_173760 ; Ensembl: ENSMUSG00000040648 ) is located on Mouse chromosome 1. 28 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 28 (Transcript: ENSMUST00000042509). Exon 3~5 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: Exon 3 starts from about 3.93% of the coding region. Exon 3~5 covers 11.01% of the coding region. The size of effective KO region: ~3479 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 3 4 5 28 Legends Exon of mouse Ppip5k2 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 3 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 676 bp section downstream of Exon 5 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(29.3% 586) | C(16.35% 327) | T(33.1% 662) | G(21.25% 425) Note: The 2000 bp section upstream of Exon 3 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(676bp) | A(25.15% 170) | C(19.38% 131) | T(32.99% 223) | G(22.49% 152) Note: The 676 bp section downstream of Exon 5 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% chr1 - 97759365 97761364 2000 browser details YourSeq 128 1533 1810 2000 85.0% chr4 + 129606050 129606206 157 browser details YourSeq 127 1686 1830 2000 95.7% chr6 + 71544792 71544939 148 browser details YourSeq 125 1686 1839 2000 92.5% chr2 - 66378468 66378632 165 browser details YourSeq 124 1676 1829 2000 93.8% chr9 + 41220831 41220986 156 browser details YourSeq 124 1686 1827 2000 95.0% chr7 + 102225405 102225547 143 browser details YourSeq 123 1686 1827 2000 95.6% chr1 - 85568369 85568512 144 browser details YourSeq 121 1686 1830 2000 94.8% chr10 + 117229485 117229631 147 browser details YourSeq 120 1686 1827 2000 93.6% chr13 - 113769241 113769384 144 browser details YourSeq 120 1686 1836 2000 91.7% chr5 + 57315211 57315362 152 browser details YourSeq 120 1686 1826 2000 94.2% chr15 + 82233611 82233752 142 browser details YourSeq 119 1672 1830 2000 87.3% chr12 + 72764505 72764656 152 browser details YourSeq 118 1686 1830 2000 93.2% chr4 + 32963061 32963204 144 browser details YourSeq 117 1686 1830 2000 92.8% chr6 - 148193122 148193267 146 browser details YourSeq 117 1688 1834 2000 92.8% chr6 - 104272376 104272525 150 browser details YourSeq 117 1683 1826 2000 91.0% chr17 - 29642787 29642927 141 browser details YourSeq 117 1679 1826 2000 93.4% chr12 - 76688932 76689102 171 browser details YourSeq 117 1692 1830 2000 94.7% chr10 - 7290741 7290881 141 browser details YourSeq 117 1686 1809 2000 97.6% chr12 + 72696179 72696304 126 browser details YourSeq 117 1672 1830 2000 92.7% chr1 + 91467197 91467357 161 Note: The 2000 bp section upstream of Exon 3 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 676 1 676 676 100.0% chr1 - 97755210 97755885 676 browser details YourSeq 56 135 281 676 90.0% chrX - 133942815 133942967 153 browser details YourSeq 54 133 436 676 78.2% chr18 + 10630228 10630518 291 browser details YourSeq 49 144 284 676 90.0% chr16 + 96843193 96843334 142 browser details YourSeq 47 133 276 676 83.7% chr12 - 55532759 55532900 142 browser details YourSeq 46 133 271 676 83.9% chr5 + 12062079 12062216 138 browser details YourSeq 45 129 192 676 80.8% chr18 - 25095576 25095635 60 browser details YourSeq 44 132 283 676 90.6% chr1 - 156316533 156316686 154 browser details YourSeq 42 135 196 676 83.9% chr1 - 89429209 89429270 62 browser details YourSeq 41 133 280 676 93.8% chr8 - 15339353 15339500 148 browser details YourSeq 41 126 179 676 88.9% chr16 - 25343737 25343791 55 browser details YourSeq 41 128 207 676 70.8% chr12 - 32455925 32455996 72 browser details YourSeq 41 136 276 676 83.4% chr11 - 6556526 6556665 140 browser details YourSeq 39 366 434 676 78.3% chr10 - 126384779 126384847 69 browser details YourSeq 39 133 283 676 90.7% chr10 - 58439550 58439699 150 browser details YourSeq 39 134 178 676 93.4% chr9 + 98719605 98719649 45 browser details YourSeq 39 133 179 676 91.5% chr3 + 94534428 94534474 47 browser details YourSeq 38 133 179 676 91.4% chr4 + 46366309 46366361 53 browser details YourSeq 37 133 281 676 95.3% chr17 + 74404242 74404390 149 browser details YourSeq 37 132 178 676 89.4% chr1 + 49843093 49843139 47 Note: The 676 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Ppip5k2 diphosphoinositol pentakisphosphate kinase 2 [ Mus musculus (house mouse) ] Gene ID: 227399, updated on 12-Aug-2019 Gene summary Official Symbol Ppip5k2 provided by MGI Official Full Name diphosphoinositol pentakisphosphate kinase 2 provided by MGI Primary source MGI:MGI:2142810 See related Ensembl:ENSMUSG00000040648 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 Vip2; Cfap160; Hisppd1; AW555814; mKIAA0433; D330021B20 Expression Ubiquitous expression in CNS E18 (RPKM 10.9), CNS E14 (RPKM 10.8) and 26 other tissues See more Orthologs human all Genomic context Location: 1; 1 D See Ppip5k2 in Genome Data Viewer Exon count: 32 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (97706043..97770411, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (99602623..99666669, complement) Chromosome 1 - NC_000067.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Ppip5k2 ENSMUSG00000040648 Description diphosphoinositol pentakisphosphate kinase 2 [Source:MGI Symbol;Acc:MGI:2142810] Gene Synonyms Cfap160, Hisppd1, Vip2 Location Chromosome 1: 97,706,048-97,770,411 reverse strand. GRCm38:CM000994.2 About this gene This gene has 7 transcripts (splice variants), 151 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ppip5k2- ENSMUST00000042509.12 5882 1129aa ENSMUSP00000043401.7 Protein coding CCDS35678 Q6ZQB6 TSL:1 201 GENCODE basic Ppip5k2- ENSMUST00000112845.7 6239 1242aa ENSMUSP00000108466.1 Protein coding - E9Q9J4 TSL:5 202 GENCODE basic APPRIS P5 Ppip5k2- ENSMUST00000171129.7 3425 1123aa ENSMUSP00000132889.1 Protein coding - Q6ZQB6 TSL:1 205 GENCODE basic APPRIS ALT1 Ppip5k2- ENSMUST00000191556.1 561 92aa ENSMUSP00000139980.1 Protein coding - A0A087WPZ7 CDS 5' incomplete 207 TSL:5 Ppip5k2- ENSMUST00000137758.1 4055 No - Retained - - TSL:1 203 protein intron Ppip5k2- ENSMUST00000152788.1 3844 No - Retained - - TSL:1 204 protein intron Ppip5k2- ENSMUST00000189992.1 2523 No - Retained - - TSL:NA 206 protein intron Page 7 of 9 https://www.alphaknockout.com 84.36 kb Forward strand 97.70Mb 97.72Mb 97.74Mb 97.76Mb 97.78Mb Genes Gin1-205 >nonsense mediated decay (Comprehensive set... Gin1-207 >nonsense mediated decay Gin1-206 >retained intron Gin1-203 >protein coding Gin1-202 >protein coding Gin1-201 >protein coding Gin1-204 >lncRNA Contigs AC099860.13 > AC162298.4 > Genes (Comprehensive set... < Ppip5k2-203retained intro<n Ppip5k2-206retained intron < Ppip5k2-204retained intron < Ppip5k2-202protein coding < Ppip5k2-201protein coding < Ppip5k2-205protein coding < Ppip5k2-207protein coding Regulatory Build 97.70Mb 97.72Mb 97.74Mb 97.76Mb 97.78Mb Reverse strand 84.36 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript
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