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Mouse Mrgpre Knockout Project (CRISPR/Cas9)

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Mouse Mrgpre Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Mrgpre Knockout Project (CRISPR/Cas9) Objective: To create a Mrgpre 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 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: Mice homozygous for a knock-out allele exhibit alterations in the development but not maintenance of allodynia. Exon 2 starts from about 0.11% of the coding region. Exon 2 covers 100.0% of the coding region. The size of effective KO region: ~928 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 Legends Exon of mouse Mrgpre Knockout region Page 2 of 8 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 start codon 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 2000 bp section downstream of stop codon is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(23.0% 460) | C(28.55% 571) | T(22.95% 459) | G(25.5% 510) Note: The 2000 bp section upstream of start codon 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(2000bp) | A(25.8% 516) | C(27.65% 553) | T(22.0% 440) | G(24.55% 491) Note: The 2000 bp section downstream of stop codon 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 8 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% chr7 - 143781765 143783764 2000 browser details YourSeq 30 302 333 2000 100.0% chr5 + 104401791 104401969 179 browser details YourSeq 28 306 334 2000 100.0% chr5 + 132670579 132670608 30 browser details YourSeq 27 300 329 2000 96.6% chr12 - 107448188 107448221 34 browser details YourSeq 27 52 81 2000 96.7% chr1 + 173811836 173811866 31 browser details YourSeq 25 306 334 2000 96.5% chr4 + 124093488 124093518 31 browser details YourSeq 24 52 78 2000 84.0% chr5 - 65183968 65183992 25 browser details YourSeq 20 302 321 2000 100.0% chr4 - 58314667 58314686 20 Note: The 2000 bp section upstream of start codon 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 2000 1 2000 2000 100.0% chr7 - 143778835 143780834 2000 browser details YourSeq 132 689 862 2000 97.3% chr1 - 72726220 72911945 185726 browser details YourSeq 95 766 893 2000 94.5% chr15 - 99380665 99380854 190 browser details YourSeq 81 611 891 2000 95.6% chr2 + 104562860 104563217 358 browser details YourSeq 80 702 891 2000 84.8% chr17 - 46584589 46584748 160 browser details YourSeq 80 666 764 2000 94.6% chr6 + 122547805 122547961 157 browser details YourSeq 79 767 860 2000 94.4% chr1 + 20761965 20762058 94 browser details YourSeq 75 775 859 2000 96.4% chr8 + 15389260 15389352 93 browser details YourSeq 73 740 845 2000 97.5% chr7 + 77540021 77540162 142 browser details YourSeq 72 610 750 2000 93.9% chr12 + 91791524 91791671 148 browser details YourSeq 68 786 862 2000 97.4% chr4 + 148166831 148166949 119 browser details YourSeq 67 698 892 2000 79.2% chr14 - 77791457 77791565 109 browser details YourSeq 66 674 749 2000 94.7% chr10 - 110925019 110925106 88 browser details YourSeq 65 679 749 2000 97.2% chr5 - 109608782 109608880 99 browser details YourSeq 62 612 716 2000 95.6% chr15 - 38133284 38133389 106 browser details YourSeq 60 710 819 2000 78.8% chr16 - 45168351 45168425 75 browser details YourSeq 59 616 737 2000 71.8% chr12 - 119026451 119026528 78 browser details YourSeq 58 611 892 2000 74.2% chr5 - 30923382 30923539 158 browser details YourSeq 58 682 744 2000 96.9% chr4 - 85152757 85152829 73 browser details YourSeq 58 610 729 2000 98.4% chr11 - 63152766 63152886 121 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 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 6 of 8 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 7 of 8 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 8 of 8.
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