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Mouse Paqr9 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Paqr9 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Paqr9 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Paqr9 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Paqr9 gene (NCBI Reference Sequence: NM_198414 ; Ensembl: ENSMUSG00000064225 ) is located on Mouse chromosome 9. 1 exon is identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000079597). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Paqr9 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-231G6 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: Exon 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~1158 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA regigoRnNA region Wildtype allele A T G T G 5' A 3' 1 Targeting vector A T G T G A Targeted allele A T G T G A Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Paqr9 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(7125bp) | A(23.78% 1694) | C(26.82% 1911) | T(25.88% 1844) | G(23.52% 1676) 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% chr9 + 95556960 95559959 3000 browser details YourSeq 74 400 519 3000 79.7% chr11 - 75218058 75218176 119 browser details YourSeq 64 199 469 3000 70.8% chr13 - 58413068 58413201 134 browser details YourSeq 63 396 521 3000 91.8% chr17 - 56769858 56770083 226 browser details YourSeq 63 402 470 3000 97.2% chr12 + 83977152 84295592 318441 browser details YourSeq 61 399 520 3000 78.8% chr12 - 76054200 76054320 121 browser details YourSeq 57 399 472 3000 89.2% chr13 - 111863689 111863763 75 browser details YourSeq 55 399 520 3000 75.8% chr1 - 101188473 101188593 121 browser details YourSeq 55 398 469 3000 88.9% chr11 + 106966601 106966673 73 browser details YourSeq 54 402 517 3000 72.3% chr9 + 120145109 120145206 98 browser details YourSeq 52 399 469 3000 90.0% chr13 - 41143875 41143944 70 browser details YourSeq 52 399 520 3000 76.4% chr11 - 88971070 88971190 121 browser details YourSeq 52 434 515 3000 81.8% chr11 - 58378084 58378165 82 browser details YourSeq 52 398 472 3000 85.4% chr2 + 170095811 170095886 76 browser details YourSeq 52 399 520 3000 94.9% chr10 + 91179334 91179788 455 browser details YourSeq 50 399 468 3000 88.6% chr17 - 78757695 78757763 69 browser details YourSeq 50 402 469 3000 92.8% chr10 - 73419019 73419085 67 browser details YourSeq 50 399 469 3000 86.0% chr1 - 72565797 72565868 72 browser details YourSeq 49 406 484 3000 81.1% chr7 + 133216990 133217068 79 browser details YourSeq 48 399 468 3000 84.3% chr12 + 71024123 71024192 70 Note: The 3000 bp section upstream of Exon 1 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% chr9 + 95561085 95564084 3000 browser details YourSeq 35 51 97 3000 79.5% chr12 + 109890018 109890059 42 browser details YourSeq 33 61 98 3000 97.2% chr17 - 59738465 59738507 43 browser details YourSeq 30 1756 1785 3000 100.0% chr2 + 58987616 58987645 30 browser details YourSeq 30 51 82 3000 90.4% chr11 + 88978645 88978675 31 browser details YourSeq 29 2429 2461 3000 96.9% chr11 + 52540372 52540408 37 browser details YourSeq 28 61 99 3000 74.2% chr2 - 5354988 5355019 32 browser details YourSeq 27 869 899 3000 96.7% chr16 + 29280122 29280154 33 browser details YourSeq 26 2405 2437 3000 78.6% chr5 + 136201613 136201641 29 browser details YourSeq 26 2405 2441 3000 93.4% chr4 + 133496561 133496597 37 browser details YourSeq 26 2405 2437 3000 96.5% chr3 + 49929933 49929977 45 browser details YourSeq 24 2406 2437 3000 75.9% chr17 + 72320195 72320223 29 browser details YourSeq 24 873 897 3000 100.0% chr14 + 21084050 21084076 27 browser details YourSeq 24 2407 2437 3000 96.2% chr11 + 29701591 29701623 33 browser details YourSeq 22 1219 1243 3000 95.9% chr3 - 104150796 104150822 27 Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Paqr9 progestin and adipoQ receptor family member IX [ Mus musculus (house mouse) ] Gene ID: 75552, updated on 12-Aug-2019 Gene summary Official Symbol Paqr9 provided by MGI Official Full Name progestin and adipoQ receptor family member IX provided by MGI Primary source MGI:MGI:1922802 See related Ensembl:ENSMUSG00000064225 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 1700020G04Rik Orthologs human all Genomic context Location: 9; 9 E3.3 See Paqr9 in Genome Data Viewer Exon count: 1 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (95559817..95562121) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (95460236..95462540) Chromosome 9 - NC_000075.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Paqr9 ENSMUSG00000064225 Description progestin and adipoQ receptor family member IX [Source:MGI Symbol;Acc:MGI:1922802] Gene Synonyms 1700020G04Rik Location Chromosome 9: 95,559,657-95,568,023 forward strand. GRCm38:CM001002.2 About this gene This gene has 1 transcript (splice variant), 163 orthologues, 10 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Paqr9-201 ENSMUST00000079597.6 8367 375aa ENSMUSP00000078547.5 Protein coding CCDS23409 Q6TCG2 TSL:NA GENCODE basic APPRIS P1 28.37 kb Forward strand 95.55Mb 95.56Mb 95.57Mb Genes (Comprehensive set... Gm32281-201 >lncRNA Paqr9-201 >protein coding Gm32281-202 >lncRNA Contigs < AC159895.2 Regulatory Build 95.55Mb 95.56Mb 95.57Mb Reverse strand 28.37 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000079597 8.37 kb Forward strand Paqr9-201 >protein coding ENSMUSP00000078... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam AdipoR/Haemolysin-III-related PANTHER AdipoR/Haemolysin-III-related PTHR20855:SF58 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 375 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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