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Mouse Mrgpre Conditional Knockout Project (CRISPR/Cas9)

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Mouse Mrgpre Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Mrgpre Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Mrgpre conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mrgpre gene (NCBI Reference Sequence: NM_175534 ; Ensembl: ENSMUSG00000048965 ) is located on Mouse chromosome 7. 2 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 2 (Transcript: ENSMUST00000054048). 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 Mrgpre gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-271L24 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 knock-out allele exhibit alterations in the development but not maintenance of allodynia. Exon 2 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 2. The size of effective cKO region: ~1252 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 A 5' gRNA region A 3' 1 2 Targeting vector T A A Targeted allele T A A Constitutive KO allele (After Cre recombination) Legends Exon of mouse Mrgpre 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(6930bp) | A(22.31% 1546) | C(29.24% 2026) | T(22.87% 1585) | G(25.58% 1773) 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% chr7 - 143781765 143784764 3000 browser details YourSeq 38 1302 1377 3000 88.4% chr12 + 5772489 5772563 75 browser details YourSeq 29 1299 1333 3000 96.9% chr10 + 12666318 12666356 39 browser details YourSeq 27 1300 1329 3000 96.6% chr12 - 107448188 107448221 34 browser details YourSeq 27 1052 1081 3000 96.7% chr1 + 173811836 173811866 31 browser details YourSeq 20 1302 1321 3000 100.0% chr4 - 58314667 58314686 20 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% chr7 - 143777835 143780834 3000 browser details YourSeq 229 644 2470 3000 95.0% chr11 + 54662117 54776243 114127 browser details YourSeq 179 618 2429 3000 94.2% chr17 - 84522835 84642118 119284 browser details YourSeq 155 674 2468 3000 94.4% chr10 - 110667275 110925106 257832 browser details YourSeq 115 2149 2519 3000 81.3% chr17 - 7750501 7750874 374 browser details YourSeq 111 2140 2519 3000 83.7% chr9 - 51037083 51037473 391 browser details YourSeq 94 2319 2510 3000 76.0% chr8 - 22993164 22993355 192 browser details YourSeq 92 2313 2514 3000 75.5% chr6 + 107854905 107855108 204 browser details YourSeq 89 2391 2599 3000 88.1% chr5 + 133598954 133599513 560 browser details YourSeq 88 2368 2519 3000 89.3% chr15 - 52611895 52612048 154 browser details YourSeq 88 2368 2517 3000 90.1% chr12 + 26419156 26502571 83416 browser details YourSeq 81 2376 2519 3000 88.0% chr5 - 37003337 37003485 149 browser details YourSeq 81 2375 2518 3000 91.0% chr5 - 30472281 30472431 151 browser details YourSeq 81 611 891 3000 95.6% chr2 + 104562860 104563217 358 browser details YourSeq 80 2368 2519 3000 85.3% chrX + 36995701 36995856 156 browser details YourSeq 79 2376 2518 3000 79.3% chr7 + 63572661 63572786 126 browser details YourSeq 79 767 860 3000 94.4% chr1 + 20761965 20762058 94 browser details YourSeq 77 2386 2516 3000 88.3% chr4 - 35589737 35589868 132 browser details YourSeq 76 2391 2510 3000 89.7% chr14 + 60276954 60277076 123 browser details YourSeq 76 2389 2519 3000 80.4% chr1 + 121413415 121413542 128 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: Mrgpre MAS-related GPR, member E [ Mus musculus (house mouse) ] Gene ID: 244238, updated on 12-Aug-2019 Gene summary Official Symbol Mrgpre provided by MGI Official Full Name MAS-related GPR, member E provided by MGI Primary source MGI:MGI:2441884 See related Ensembl:ENSMUSG00000048965 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 Mgrf; MrgE; Ebrt3; C130069N09Rik Expression Ubiquitous expression in thymus adult (RPKM 3.2), CNS E18 (RPKM 3.1) and 26 other tissues See more Orthologs human all Genomic context Location: 7; 7 F5 See Mrgpre in Genome Data Viewer Exon count: 2 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (143778363..143784500, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (150964268..150970405, complement) Chromosome 7 - NC_000073.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Mrgpre ENSMUSG00000048965 Description MAS-related GPR, member E [Source:MGI Symbol;Acc:MGI:2441884] Gene Synonyms C130069N09Rik, MrgE Location Chromosome 7: 143,778,363-143,784,500 reverse strand. GRCm38:CM001000.2 About this gene This gene has 1 transcript (splice variant), 51 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 Mrgpre-201 ENSMUST00000054048.9 3761 310aa ENSMUSP00000058000.8 Protein coding CCDS22046 Q4V9R2 Q91ZB7 TSL:1 GENCODE basic APPRIS P1 26.14 kb Forward strand 143.77Mb 143.78Mb 143.79Mb Genes Gm22064-201 >miRNA (Comprehensive set... Contigs AC158299.7 > Genes (Comprehensive set... < Gm44998-201TEC < Mrgpre-201protein coding Regulatory Build 143.77Mb 143.78Mb 143.79Mb Reverse strand 26.14 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000054048 < Mrgpre-201protein coding Reverse strand 6.14 kb ENSMUSP00000058... Transmembrane heli... Low complexity (Seg) Superfamily SSF81321 Prints Mas-related G protein-coupled receptor E Mas-related G protein-coupled receptor family G protein-coupled receptor, rhodopsin-like PROSITE profiles GPCR, rhodopsin-like, 7TM PANTHER Mas-related G protein-coupled receptor family Mas-related G protein-coupled receptor E Gene3D 1.20.1070.10 CDD cd15112 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 310 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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