https://www.alphaknockout.com Mouse Mrgprg Knockout Project (CRISPR/Cas9) Objective: To create a Mrgprg knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mrgprg gene (NCBI Reference Sequence: NM_203492 ; Ensembl: ENSMUSG00000050276 ) is located on Mouse chromosome 7. 2 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 2 (Transcript: ENSMUST00000058092). 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: Exon 2 starts from about 0.12% of the coding region. Exon 2 covers 100.0% of the coding region. The size of effective KO region: ~865 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 Mrgprg 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. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. 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(23.6% 472) | C(26.6% 532) | T(23.9% 478) | G(25.9% 518) 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(26.0% 520) | T(22.8% 456) | G(25.4% 508) 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 - 143765374 143767373 2000 browser details YourSeq 35 80 150 2000 89.8% chr2 - 37958161 37958230 70 browser details YourSeq 34 132 190 2000 76.8% chr7 + 98332661 98332718 58 browser details YourSeq 33 88 152 2000 92.4% chr8 + 84468181 84468245 65 browser details YourSeq 31 139 197 2000 91.0% chr17 + 93340279 93340336 58 browser details YourSeq 29 139 197 2000 74.6% chr17 - 47257701 47257759 59 browser details YourSeq 29 1225 1255 2000 96.8% chr7 + 63365283 63365313 31 browser details YourSeq 29 139 197 2000 91.5% chr2 + 120547831 120547890 60 browser details YourSeq 29 139 197 2000 74.6% chr1 + 82760758 82760816 59 browser details YourSeq 27 1229 1255 2000 100.0% chr2 - 149791130 149791156 27 browser details YourSeq 27 1227 1259 2000 87.1% chr11 + 110902100 110902131 32 browser details YourSeq 26 1230 1255 2000 100.0% chr3 - 54548828 54548853 26 browser details YourSeq 24 138 161 2000 100.0% chr7 - 132677146 132677169 24 browser details YourSeq 24 88 111 2000 100.0% chr4 - 150609599 150609622 24 browser details YourSeq 24 113 152 2000 80.0% chr2 - 161091879 161091918 40 browser details YourSeq 23 99 125 2000 92.6% chr9 + 7083590 7083616 27 browser details YourSeq 23 72 94 2000 100.0% chr2 + 3773875 3773897 23 browser details YourSeq 23 138 160 2000 100.0% chr18 + 63396574 63396596 23 browser details YourSeq 22 177 198 2000 100.0% chr3 - 102209251 102209272 22 browser details YourSeq 22 474 509 2000 80.6% chr2 + 60414836 60414871 36 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 - 143762507 143764506 2000 browser details YourSeq 65 815 943 2000 78.1% chr13 - 65134722 65134858 137 browser details YourSeq 65 836 941 2000 81.2% chr6 + 97333823 97334228 406 browser details YourSeq 64 835 943 2000 77.9% chr18 - 36815948 36816054 107 browser details YourSeq 62 835 943 2000 77.0% chr8 - 87923972 87924078 107 browser details YourSeq 60 835 930 2000 81.3% chr1 - 156538605 156538700 96 browser details YourSeq 60 835 930 2000 80.5% chr2 + 163470881 163470975 95 browser details YourSeq 59 838 943 2000 76.3% chr13 - 63344248 63344351 104 browser details YourSeq 59 828 928 2000 79.8% chr13 - 30191910 30192015 106 browser details YourSeq 59 834 930 2000 80.5% chr1 - 192556788 192556884 97 browser details YourSeq 59 835 940 2000 78.4% chr7 + 144568000 144568448 449 browser details YourSeq 59 835 943 2000 77.5% chr15 + 80986138 80986243 106 browser details YourSeq 58 835 952 2000 83.8% chr17 - 47103184 47103319 136 browser details YourSeq 58 835 930 2000 80.3% chr11 - 20546585 20546680 96 browser details YourSeq 58 835 930 2000 80.3% chr11 + 51769934 51770029 96 browser details YourSeq 57 830 927 2000 76.1% chr4 - 72717354 72717449 96 browser details YourSeq 57 847 942 2000 76.6% chr3 - 144649784 144649877 94 browser details YourSeq 57 846 930 2000 96.9% chr2 - 147009543 147009908 366 browser details YourSeq 56 835 944 2000 86.9% chr6 - 85936336 85936454 119 browser details YourSeq 56 835 943 2000 74.1% chr5 - 143010418 143010524 107 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: Mrgprg MAS-related GPR, member G [ Mus musculus (house mouse) ] Gene ID: 381974, updated on 12-Aug-2019 Gene summary Official Symbol Mrgprg provided by MGI Official Full Name MAS-related GPR, member G provided by MGI Primary source MGI:MGI:3033145 See related Ensembl:ENSMUSG00000050276 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 Mrgg; Gm1098 Expression Biased expression in mammary gland adult (RPKM 5.3), placenta adult (RPKM 1.6) and 4 other tissues See more Orthologs human all Genomic context Location: 7; 7 F5 See Mrgprg in Genome Data Viewer Exon count: 2 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (143763710..143766993, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (150949615..150952898, complement) Chromosome 7 - NC_000073.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Mrgprg ENSMUSG00000050276 Description MAS-related GPR, member G [Source:MGI Symbol;Acc:MGI:3033145] Gene Synonyms LOC381974, MrgG Location Chromosome 7: 143,763,710-143,766,993 reverse strand. GRCm38:CM001000.2 About this gene This gene has 2 transcripts (splice variants), 91 orthologues, 21 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Mrgprg-201 ENSMUST00000058092.7 1821 289aa ENSMUSP00000060411.6 Protein coding CCDS22045 Q91ZB5 TSL:1 GENCODE basic APPRIS P1 Mrgprg-202 ENSMUST00000208986.1 671 156aa ENSMUSP00000146374.1 Protein coding - A0A140LHD7 CDS 3' incomplete TSL:NA 23.28 kb Forward strand 143.755Mb 143.760Mb 143.765Mb 143.770Mb 143.775Mb Contigs AC158299.7 > Genes (Comprehensive set... < Osbpl5-203protein coding < Mrgprg-201protein coding < Gm44998-201TEC < Mrgprg-202protein coding Regulatory Build 143.755Mb 143.760Mb 143.765Mb 143.770Mb 143.775Mb Reverse strand 23.28 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 processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000058092 < Mrgprg-201protein coding Reverse strand 3.28 kb ENSMUSP00000060... Transmembrane heli... Low complexity (Seg) Superfamily SSF81321 Prints G protein-coupled receptor, rhodopsin-like Mas-related G protein-coupled receptor family PANTHER Mas-related G protein-coupled receptor family Mas-related G protein-coupled receptor G Gene3D 1.20.1070.10 CDD Mas-related G protein-coupled receptor G All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 289 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.