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Mouse Rgs16 Knockout Project (CRISPR/Cas9)

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Mouse Rgs16 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rgs16 Knockout Project (CRISPR/Cas9) Objective: To create a Rgs16 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rgs16 gene (NCBI Reference Sequence: NM_011267 ; Ensembl: ENSMUSG00000026475 ) is located on Mouse chromosome 1. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000027748). Exon 1~5 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 increased fatty acid oxidation and circulating ketone levels when fed a high-fat diet. Mice homozygous for a different knock-out allele exhibit impaired Th1 and Th2 chemotaxis and increased susceptibility toparasitic infection. Exon 1 starts from about 0.17% of the coding region. Exon 1~5 covers 100.0% of the coding region. The size of effective KO region: ~3384 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 3 4 5 Legends Exon of mouse Rgs16 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. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. 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(20.1% 402) | C(31.6% 632) | T(27.85% 557) | G(20.45% 409) Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(23.25% 465) | C(23.45% 469) | T(24.45% 489) | G(28.85% 577) 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% chr1 + 153738470 153740469 2000 browser details YourSeq 139 11 187 2000 90.3% chr5 + 144084640 144084879 240 browser details YourSeq 136 1 211 2000 89.7% chr10 - 91263634 91263842 209 browser details YourSeq 135 1 180 2000 90.0% chr10 - 76175500 76175698 199 browser details YourSeq 131 1 249 2000 82.4% chr13 - 93511673 93511907 235 browser details YourSeq 130 2 211 2000 85.7% chr5 - 122148147 122148334 188 browser details YourSeq 128 1 180 2000 90.7% chr1 + 136193467 136193681 215 browser details YourSeq 126 1 211 2000 81.3% chr19 + 25375955 25376141 187 browser details YourSeq 125 1 171 2000 94.6% chr9 - 43268313 43392363 124051 browser details YourSeq 123 1 174 2000 91.1% chr9 + 75956509 75957095 587 browser details YourSeq 119 1 181 2000 92.9% chr6 - 51620989 51621234 246 browser details YourSeq 119 19 211 2000 88.5% chr11 + 106358406 106358610 205 browser details YourSeq 118 1 190 2000 83.2% chr2 - 148448957 148449125 169 browser details YourSeq 117 1 180 2000 85.2% chrX + 95674881 95675043 163 browser details YourSeq 117 1 180 2000 83.8% chr14 + 78290591 78290751 161 browser details YourSeq 116 1 211 2000 92.7% chr10 - 39573744 39574103 360 browser details YourSeq 115 5 183 2000 82.3% chr11 + 99095444 99095605 162 browser details YourSeq 115 11 211 2000 82.9% chr1 + 161206849 161207017 169 browser details YourSeq 114 1 151 2000 90.8% chr10 - 6008641 6008845 205 browser details YourSeq 113 1 173 2000 91.3% chr16 + 23611303 23611495 193 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% chr1 + 153743854 153745853 2000 browser details YourSeq 36 1959 2000 2000 84.7% chr14 - 87408198 87408236 39 browser details YourSeq 32 1961 2000 2000 91.2% chr12 - 24496784 24496822 39 browser details YourSeq 31 1967 2000 2000 97.1% chr10 - 4258980 4259020 41 browser details YourSeq 30 1961 2000 2000 97.0% chr8 - 88722195 88722234 40 browser details YourSeq 30 1968 2000 2000 97.0% chr2 - 119881149 119881184 36 browser details YourSeq 30 1968 2000 2000 97.0% chr16 - 17275260 17275299 40 browser details YourSeq 29 1970 2000 2000 90.0% chr9 - 3743338 3743367 30 browser details YourSeq 28 1967 2000 2000 90.0% chr15 - 79987743 79987775 33 browser details YourSeq 28 1965 2000 2000 96.7% chr14 - 80716280 80716317 38 browser details YourSeq 28 1964 2000 2000 96.7% chr16 + 16837266 16837311 46 browser details YourSeq 28 1973 2000 2000 100.0% chr12 + 84922881 84922908 28 browser details YourSeq 28 1970 2000 2000 86.3% chr10 + 31279139 31279167 29 browser details YourSeq 27 1974 2000 2000 100.0% chr7 + 123153640 123153666 27 browser details YourSeq 27 1974 2000 2000 100.0% chr17 + 21735842 21735868 27 browser details YourSeq 27 1974 2000 2000 100.0% chr16 + 36397267 36397293 27 browser details YourSeq 27 1974 2000 2000 100.0% chr15 + 41553808 41553834 27 browser details YourSeq 26 1973 2000 2000 88.9% chr5 - 147459175 147459201 27 browser details YourSeq 26 1973 2000 2000 96.5% chr4 - 137821734 137821761 28 browser details YourSeq 26 1975 2000 2000 100.0% chr10 - 69828320 69828345 26 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: Rgs16 regulator of G-protein signaling 16 [ Mus musculus (house mouse) ] Gene ID: 19734, updated on 12-Aug-2019 Gene summary Official Symbol Rgs16 provided by MGI Official Full Name regulator of G-protein signaling 16 provided by MGI Primary source MGI:MGI:108407 See related Ensembl:ENSMUSG00000026475 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 Rgsr; Rgs14 Expression Broad expression in liver E18 (RPKM 12.8), CNS E11.5 (RPKM 10.4) and 15 other tissues See more Orthologs human all Genomic context Location: 1 G3; 1 65.43 cM See Rgs16 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (153740353..153745468) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (155587483..155592598) Chromosome 1 - NC_000067.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Rgs16 ENSMUSG00000026475 Description regulator of G-protein signaling 16 [Source:MGI Symbol;Acc:MGI:108407] Gene Synonyms Rgsr Location Chromosome 1: 153,740,349-153,745,468 forward strand. GRCm38:CM000994.2 About this gene This gene has 1 transcript (splice variant), 161 orthologues, 23 paralogues, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rgs16-201 ENSMUST00000027748.7 2339 201aa ENSMUSP00000027748.7 Protein coding CCDS15376 P97428 Q542U0 TSL:1 GENCODE basic APPRIS P1 25.12 kb Forward strand 153.735Mb 153.740Mb 153.745Mb 153.750Mb 153.755Mb Genes (Comprehensive set... Rgs16-201 >protein coding Rnasel-201 >protein coding Rnasel-205 >protein coding Gm28512-201 >lncRNA Rnasel-206 >protein coding Rnasel-203 >retained intron Rnasel-204 >protein coding Contigs AC154140.7 > Genes < C230024C17Rik-201lncRNA < Gm28513-201lncRNA (Comprehensive set... Regulatory Build 153.735Mb 153.740Mb 153.745Mb 153.750Mb 153.755Mb Reverse strand 25.12 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000027748 5.12 kb Forward strand Rgs16-201 >protein coding ENSMUSP00000027... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Superfamily RGS domain superfamily SMART RGS domain Prints RGS domain Pfam RGS domain PROSITE profiles RGS domain PANTHER PTHR10845:SF187 PTHR10845 Gene3D RGS, subdomain 1/3 1.10.167.10 CDD cd08710 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 201 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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