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Mouse Tiparp Conditional Knockout Project (CRISPR/Cas9)

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Mouse Tiparp Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Tiparp Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Tiparp conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tiparp gene (NCBI Reference Sequence: NM_178892 ; Ensembl: ENSMUSG00000034640 ) is located on Mouse chromosome 3. 6 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000047906). 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 Tiparp gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-371A15 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: Mice homozygous for a gene trapped allele exhibit postnatal lethality, skeletal and craniofacial defects, kidney defects and embryonic hemorrhaging. Exon 2 starts from about 0.1% 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: 2591 bp, and the size of intron 2 for 3'-loxP site insertion: 14022 bp. The size of effective cKO region: ~1420 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Tiparp Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com 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(7920bp) | A(26.65% 2111) | C(20.72% 1641) | T(29.13% 2307) | G(23.5% 1861) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN -------------------------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr3 + 65528016 65531015 3000 browser details YourSeq 49 1577 1701 3000 92.9% chr7 + 4507990 4508119 130 browser details YourSeq 49 1577 1700 3000 81.9% chr18 + 67836963 67837077 115 browser details YourSeq 48 1577 1700 3000 68.7% chr9 - 85873672 85873722 51 browser details YourSeq 46 1577 1628 3000 96.1% chr9 - 121128070 121128237 168 browser details YourSeq 46 1577 1629 3000 94.3% chr19 + 53349444 53349500 57 browser details YourSeq 45 1577 1628 3000 96.0% chr7 + 74346140 74346193 54 browser details YourSeq 43 1578 1628 3000 93.9% chr6 - 147347515 147347565 51 browser details YourSeq 43 1577 1628 3000 95.8% chr9 + 106382632 106382685 54 browser details YourSeq 43 1590 1700 3000 74.6% chr17 + 4041414 4041507 94 browser details YourSeq 42 1580 1628 3000 87.3% chr7 - 3388154 3388200 47 browser details YourSeq 42 1577 1628 3000 95.7% chr2 - 169478102 169478283 182 browser details YourSeq 42 1577 1628 3000 84.5% chr12 - 24676910 24676957 48 browser details YourSeq 41 1580 1626 3000 95.5% chr5 - 136129848 136129898 51 browser details YourSeq 41 1580 1626 3000 95.5% chr4 - 53165561 53165611 51 browser details YourSeq 41 1577 1626 3000 95.6% chr11 - 79791569 79791626 58 browser details YourSeq 41 1577 1626 3000 93.7% chr6 + 28531734 28531799 66 browser details YourSeq 40 1590 1700 3000 66.7% chr3 - 97191501 97191564 64 browser details YourSeq 40 1577 1626 3000 78.6% chr13 - 95248980 95249021 42 browser details YourSeq 40 1577 1628 3000 93.4% chr10 + 68225212 68225263 52 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% chr3 + 65532436 65535435 3000 browser details YourSeq 152 105 644 3000 85.1% chr4 + 138248264 138248638 375 browser details YourSeq 145 502 663 3000 95.7% chr7 - 101544875 101545055 181 browser details YourSeq 145 498 663 3000 92.0% chr10 + 79825325 79825487 163 browser details YourSeq 141 498 665 3000 92.7% chr10 - 88248938 88249108 171 browser details YourSeq 140 501 656 3000 92.2% chr11 - 95409711 95409863 153 browser details YourSeq 140 498 653 3000 93.6% chr19 + 48186204 48186358 155 browser details YourSeq 139 498 656 3000 94.9% chr6 - 54520294 54520463 170 browser details YourSeq 139 498 658 3000 91.9% chr5 + 36291594 36291753 160 browser details YourSeq 139 502 665 3000 90.2% chr11 + 86678277 86678429 153 browser details YourSeq 138 501 663 3000 94.9% chr4 - 108169206 108169371 166 browser details YourSeq 137 501 663 3000 94.8% chr4 + 11312801 11312964 164 browser details YourSeq 136 498 657 3000 94.8% chrX - 100841386 100841549 164 browser details YourSeq 135 494 669 3000 96.0% chr17 + 6710505 6710698 194 browser details YourSeq 134 502 645 3000 94.4% chr18 - 7349390 7349531 142 browser details YourSeq 134 498 647 3000 92.6% chr19 + 24155264 24155411 148 browser details YourSeq 133 505 652 3000 96.0% chr3 + 146128453 146128608 156 browser details YourSeq 131 498 653 3000 92.9% chr5 + 137774527 137774697 171 browser details YourSeq 130 498 648 3000 91.2% chr17 - 31213231 31213378 148 browser details YourSeq 130 501 659 3000 89.0% chr1 - 16888368 16888512 145 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Tiparp TCDD-inducible poly(ADP-ribose) polymerase [ Mus musculus (house mouse) ] Gene ID: 99929, updated on 14-Oct-2019 Gene summary Official Symbol Tiparp provided by MGI Official Full Name TCDD-inducible poly(ADP-ribose) polymerase provided by MGI Primary source MGI:MGI:2159210 See related Ensembl:ENSMUSG00000034640 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as PARP7; ARTD14; AW558171 Expression Ubiquitous expression in bladder adult (RPKM 8.4), genital fat pad adult (RPKM 4.9) and 28 other tissues See more Orthologs human all Genomic context Location: 3; 3 E1 See Tiparp in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (65528447..65555518) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (65332369..65359440) Chromosome 3 - NC_000069.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Tiparp ENSMUSG00000034640 Description TCDD-inducible poly(ADP-ribose) polymerase [Source:MGI Symbol;Acc:MGI:2159210] Gene Synonyms DDF1, PARP-7, PARP7 Location Chromosome 3: 65,528,410-65,555,518 forward strand. GRCm38:CM000996.2 About this gene This gene has 3 transcripts (splice variants), 201 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 55 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tiparp-201 ENSMUST00000047906.9 4191 657aa ENSMUSP00000048051.3 Protein coding CCDS17388 Q8C1B2 TSL:1 GENCODE basic APPRIS P1 Tiparp-202 ENSMUST00000130705.1 827 205aa ENSMUSP00000119951.1 Protein coding - D3Z7U2 CDS 3' incomplete TSL:3 Tiparp-203 ENSMUST00000154094.1 594 No protein - lncRNA - - TSL:2 47.11 kb Forward strand 65.52Mb 65.53Mb 65.54Mb 65.55Mb 65.56Mb Genes (Comprehensive set... Gm22279-201 >snRNA Tiparp-203 >lncRNA Tiparp-201 >protein coding Tiparp-202 >protein coding Contigs AC113279.7 > Genes < 4931440P22Rik-202lncRNA (Comprehensive set... < 4931440P22Rik-203lncRNA < 4931440P22Rik-204lncRNA < 4931440P22Rik-201lncRNA Regulatory Build 65.52Mb 65.53Mb 65.54Mb 65.55Mb 65.56Mb Reverse strand 47.11 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 Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000047906 27.11 kb Forward strand Tiparp-201 >protein coding ENSMUSP00000048... MobiDB lite Superfamily WWE domain superfamily SSF56399 Pfam Poly(ADP-ribose) polymerase, catalytic domain PROSITE profiles WWE domain Poly(ADP-ribose) polymerase, catalytic domain Zinc finger, CCCH-type PANTHER PTHR45740:SF7 PTHR45740 Gene3D 3.90.228.10 CDD cd01439 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 657 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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