https://www.alphaknockout.com Mouse Paqr8 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Paqr8 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Paqr8 gene (NCBI Reference Sequence: NM_028829 ; Ensembl: ENSMUSG00000025931 ) is located on Mouse chromosome 1. 3 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000189400). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Paqr8 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-191A12 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 3 covers 100.0% of the coding region. Start codon is in exon 3, and stop codon is in exon 3. The size of effective cKO region: ~1377 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 region Wildtype allele T gRNA region G 5' A 3' 1 3 Targeting vector T G A Targeted allele T G A Constitutive KO allele (After Cre recombination) Legends Exon of mouse Paqr8 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7062bp) | A(26.18% 1849) | C(22.9% 1617) | T(27.81% 1964) | G(23.11% 1632) Note: The sequence of homologous arms and cKO region 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 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% chr1 + 20931625 20934624 3000 browser details YourSeq 186 1476 2412 3000 93.5% chr17 - 71340691 71586837 246147 browser details YourSeq 139 1468 1632 3000 90.2% chr10 + 117385922 117386083 162 browser details YourSeq 135 1472 1616 3000 96.6% chr15 + 78810619 78810763 145 browser details YourSeq 134 1474 1611 3000 98.6% chr18 + 46491010 46491147 138 browser details YourSeq 133 1460 1613 3000 94.6% chr1 + 45856818 45856972 155 browser details YourSeq 132 1461 1612 3000 95.2% chr15 - 83374476 83374629 154 browser details YourSeq 132 1476 1611 3000 98.6% chr12 - 16697801 16697936 136 browser details YourSeq 130 1479 1614 3000 97.8% chr16 + 4561257 4561392 136 browser details YourSeq 130 1475 1614 3000 96.5% chr1 + 55145899 55146038 140 browser details YourSeq 129 1475 1613 3000 96.5% chr18 + 35786696 35786834 139 browser details YourSeq 129 1484 1630 3000 93.9% chr14 + 25825424 25825570 147 browser details YourSeq 126 1475 1610 3000 96.4% chr10 - 90856147 90856282 136 browser details YourSeq 126 1475 1610 3000 96.4% chr13 + 99682191 99682326 136 browser details YourSeq 126 1484 1693 3000 95.0% chr1 + 139458688 139458898 211 browser details YourSeq 125 1096 1612 3000 82.1% chr17 - 26851571 26851875 305 browser details YourSeq 125 1476 1612 3000 95.7% chr13 + 64269725 64269861 137 browser details YourSeq 124 1480 1612 3000 93.8% chr15 - 78924746 78924872 127 browser details YourSeq 124 1485 1616 3000 97.0% chr12 - 110482267 110482398 132 browser details YourSeq 123 1484 1616 3000 96.3% chr16 + 38848669 38848801 133 Note: The 3000 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 3000 1 3000 3000 100.0% chr1 + 20935687 20938686 3000 browser details YourSeq 49 2258 2360 3000 70.8% chr16 + 18879855 18879946 92 browser details YourSeq 46 217 297 3000 87.5% chr14 - 56588970 56589049 80 browser details YourSeq 46 2238 2337 3000 76.4% chr11 - 44709280 44709376 97 browser details YourSeq 39 228 286 3000 83.1% chr7 - 46842612 46842670 59 browser details YourSeq 38 2250 2302 3000 76.6% chr6 - 116064111 116064157 47 browser details YourSeq 38 226 285 3000 81.7% chr16 - 21990442 21990501 60 browser details YourSeq 36 226 281 3000 82.2% chr1 + 154593596 154593651 56 browser details YourSeq 33 230 278 3000 83.7% chr11 - 95613063 95613111 49 browser details YourSeq 33 219 257 3000 92.4% chr10 - 50587598 50587636 39 browser details YourSeq 33 2228 2267 3000 79.5% chr9 + 95607774 95607807 34 browser details YourSeq 33 228 272 3000 86.7% chr9 + 43383476 43383520 45 browser details YourSeq 33 265 297 3000 100.0% chr4 + 134446965 134446997 33 browser details YourSeq 32 2246 2287 3000 88.1% chrX + 21374315 21374356 42 browser details YourSeq 32 221 258 3000 92.2% chr2 + 26067869 26067906 38 browser details YourSeq 32 2241 2339 3000 63.2% chr14 + 56625836 56625894 59 browser details YourSeq 32 2238 2286 3000 81.0% chr13 + 46660000 46660046 47 browser details YourSeq 30 2249 2286 3000 89.5% chr2 - 121816756 121816793 38 browser details YourSeq 30 128 277 3000 96.9% chr14 + 121990539 121990688 150 browser details YourSeq 29 2247 2286 3000 79.0% chr11 - 78440486 78440523 38 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Paqr8 progestin and adipoQ receptor family member VIII [ Mus musculus (house mouse) ] Gene ID: 74229, updated on 14-Aug-2019 Gene summary Official Symbol Paqr8 provided by MGI Official Full Name progestin and adipoQ receptor family member VIII provided by MGI Primary source MGI:MGI:1921479 See related Ensembl:ENSMUSG00000025931 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 1700019B16Rik; 3110001D06Rik Expression Broad expression in cerebellum adult (RPKM 28.5), adrenal adult (RPKM 18.6) and 15 other tissues See more Orthologs human all Genomic context Location: 1; 1 A4 See Paqr8 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (20890548..20939650) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (20880703..20928837) Chromosome 1 - NC_000067.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Paqr8 ENSMUSG00000025931 Description progestin and adipoQ receptor family member VIII [Source:MGI Symbol;Acc:MGI:1921479] Gene Synonyms 1700019B16Rik, 3110001D06Rik Location Chromosome 1: 20,890,606-20,939,650 forward strand. GRCm38:CM000994.2 About this gene This gene has 4 transcripts (splice variants), 224 orthologues, 10 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Paqr8-204 ENSMUST00000189400.6 5330 354aa ENSMUSP00000141054.1 Protein coding CCDS14844 Q80ZE5 TSL:1 GENCODE basic APPRIS P1 Paqr8-201 ENSMUST00000068880.13 4704 354aa ENSMUSP00000069127.7 Protein coding CCDS14844 Q80ZE5 TSL:1 GENCODE basic APPRIS P1 Paqr8-202 ENSMUST00000167119.7 1472 354aa ENSMUSP00000128781.1 Protein coding CCDS14844 Q80ZE5 TSL:1 GENCODE basic APPRIS P1 Paqr8-203 ENSMUST00000187651.1 1463 354aa ENSMUSP00000140913.1 Protein coding CCDS14844 Q80ZE5 TSL:5 GENCODE basic APPRIS P1 69.05 kb Forward strand 20.89Mb 20.90Mb 20.91Mb 20.92Mb 20.93Mb 20.94Mb Genes (Comprehensive set... Paqr8-204 >protein coding Paqr8-201 >protein coding Paqr8-202 >protein coding Gm28064-201 >processed pseudogene Paqr8-203 >protein coding Contigs < AC156980.9 Genes < 6720483E21Rik-201lncRNA < Gm24171-201snoRNA (Comprehensive set... Regulatory Build 20.89Mb 20.90Mb 20.91Mb 20.92Mb 20.93Mb 20.94Mb Reverse strand 69.05 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 RNA gene pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000189400 49.05 kb Forward strand Paqr8-204 >protein coding ENSMUSP00000141... Transmembrane heli... Low complexity (Seg) Coiled-coils (Ncoils) Pfam AdipoR/Haemolysin-III-related PANTHER PTHR20855:SF22 AdipoR/Haemolysin-III-related All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 354 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.