https://www.alphaknockout.com Mouse Atp6v0a2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Atp6v0a2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Atp6v0a2 gene (NCBI Reference Sequence: NM_011596 ; Ensembl: ENSMUSG00000038023 ) is located on Mouse chromosome 5. 20 exons are identified, with the GTG start codon in exon 1 and the TGA stop codon in exon 20 (Transcript: ENSMUST00000037865). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Atp6v0a2 gene. 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 5 starts from about 16.86% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 655 bp, and the size of intron 5 for 3'-loxP site insertion: 2072 bp. The size of effective cKO region: ~589 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 5 6 20 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Atp6v0a2 Homology arm cKO region loxP site Page 2 of 8 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. 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7089bp) | A(19.76% 1401) | C(24.67% 1749) | T(29.48% 2090) | G(26.08% 1849) 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 8 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% chr5 + 124635946 124638945 3000 browser details YourSeq 228 2203 2764 3000 94.6% chr4 - 40212646 40714760 502115 browser details YourSeq 212 2218 2708 3000 87.2% chr9 - 113890600 113891078 479 browser details YourSeq 198 2201 2668 3000 90.0% chr2 - 29106849 29107381 533 browser details YourSeq 191 2207 2744 3000 84.6% chr4 + 127248619 127248943 325 browser details YourSeq 176 2223 2747 3000 82.6% chr11 + 78361861 78362286 426 browser details YourSeq 175 2197 2515 3000 89.0% chr9 - 21691574 21691901 328 browser details YourSeq 168 2206 2751 3000 92.4% chr2 - 146891802 146892438 637 browser details YourSeq 163 2206 2482 3000 91.1% chr11 + 115636112 115636375 264 browser details YourSeq 155 2182 2401 3000 88.0% chrX - 153291037 153291255 219 browser details YourSeq 152 2235 2630 3000 81.5% chr1 - 181739567 181739771 205 browser details YourSeq 152 2197 2365 3000 95.3% chr8 + 111647703 111647873 171 browser details YourSeq 149 2175 2365 3000 88.8% chr17 + 69393532 69393717 186 browser details YourSeq 148 2277 2765 3000 88.5% chr13 - 93798367 93799052 686 browser details YourSeq 148 2199 2392 3000 87.5% chr2 + 25154856 25155036 181 browser details YourSeq 147 2197 2365 3000 92.3% chr11 + 6379714 6379881 168 browser details YourSeq 145 1956 2365 3000 89.1% chr4 - 123064698 123065093 396 browser details YourSeq 145 2198 2365 3000 93.5% chr8 + 107499305 107499473 169 browser details YourSeq 145 2199 2369 3000 92.4% chr1 + 10107741 10107911 171 browser details YourSeq 144 2203 2364 3000 94.5% chr2 + 166013952 166014113 162 Note: The 3000 bp section upstream of Exon 5 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% chr5 + 124639535 124642534 3000 browser details YourSeq 193 838 1097 3000 94.6% chr11 + 104488231 104488861 631 browser details YourSeq 176 473 1067 3000 87.1% chr8 + 117520355 117520631 277 browser details YourSeq 172 323 1070 3000 91.7% chr10 - 52224801 52285716 60916 browser details YourSeq 171 859 1063 3000 93.6% chr4 - 107242296 107243240 945 browser details YourSeq 161 732 1067 3000 93.2% chr2 + 154475097 154475656 560 browser details YourSeq 156 871 1071 3000 92.9% chr12 + 83600799 83601241 443 browser details YourSeq 156 817 1067 3000 93.9% chr11 + 115725283 115772603 47321 browser details YourSeq 155 901 1076 3000 94.3% chr17 - 4904393 4904819 427 browser details YourSeq 154 638 1065 3000 85.3% chr17 + 80780864 80781145 282 browser details YourSeq 153 898 1069 3000 95.9% chr4 - 46902371 46902550 180 browser details YourSeq 153 816 1066 3000 94.3% chr8 + 123108427 123108682 256 browser details YourSeq 152 471 1067 3000 85.4% chr13 - 38583249 38583649 401 browser details YourSeq 151 902 1067 3000 96.4% chr5 - 23647150 23647320 171 browser details YourSeq 150 903 1085 3000 90.8% chr9 - 9031905 9032082 178 browser details YourSeq 150 901 1079 3000 91.3% chr18 + 80949776 80949950 175 browser details YourSeq 149 901 1067 3000 95.8% chr4 + 98942962 98943144 183 browser details YourSeq 148 902 1063 3000 96.3% chrX - 157463349 157463516 168 browser details YourSeq 148 902 1067 3000 95.2% chr3 - 9536124 9536304 181 browser details YourSeq 148 904 1068 3000 93.9% chr11 - 43722470 43722632 163 Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Atp6v0a2 ATPase, H+ transporting, lysosomal V0 subunit A2 [ Mus musculus (house mouse) ] Gene ID: 21871, updated on 10-Oct-2019 Gene summary Official Symbol Atp6v0a2 provided by MGI Official Full Name ATPase, H+ transporting, lysosomal V0 subunit A2 provided by MGI Primary source MGI:MGI:104855 See related Ensembl:ENSMUSG00000038023 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as ISF; Tj6; J6B7; SHIF; Stv1; TJ6M; TJ6s; ATP6a2; Atp6n2; C76904; Atp6n1d; AI385560; AW489264; V-ATPase a2; Summary 8430408C20Rik; V-ATPase 116 kDa Annotation This gene encodes a subunit of vacuolar ATPase, a multimeric enzyme that localizes to intracellular vesicles and to the information plasma membrane of specialized cells. The encoded protein is a component of the V(0) domain, which functions in proton translocation across membranes. Function of this gene is important in fetal-specific immune suppression during pregnancy. [provided by RefSeq, May 2013] Expression Annotation category: spans an assembly gap Orthologs Ubiquitous expression in large intestine adult (RPKM 48.7), duodenum adult (RPKM 33.8) and 28 other tissues See more human all Genomic context Location: 5; 5 F See Atp6v0a2 in Genome Data Viewer Exon count: 20 Annotation Status Assembly Chr Location release GRCm38.p6 108 current 5 NC_000071.6 (124629052..124724455) (GCF_000001635.26) previous MGSCv37 NC_000071.5 (125079648..125097437) , Build 37.2 5 assembly (GCF_000001635.18) (125192573..125202524) Chromosome 5 - NC_000071.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Atp6v0a2 ENSMUSG00000038023 Description ATPase, H+ transporting, lysosomal V0 subunit A2 [Source:MGI Symbol;Acc:MGI:104855] Gene Synonyms 8430408C20Rik, ATP6a2, Atp6n2, TJ6s, Tj6, V-ATPase a2 Location Chromosome 5: 124,628,576-124,724,455 forward strand. GRCm38:CM000998.2 About this gene This gene has 7 transcripts (splice variants), 278 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Atp6v0a2- ENSMUST00000037865.12 5345 856aa ENSMUSP00000039737.8 Protein coding CCDS84963 P15920 TSL:1 201 GENCODE basic APPRIS P1 Atp6v0a2- ENSMUST00000198382.4 3526 182aa ENSMUSP00000143284.1 Nonsense mediated - A0A0G2JFS2 TSL:2 205 decay Atp6v0a2- ENSMUST00000197161.1 660 83aa ENSMUSP00000143461.1 Nonsense mediated - A0A0G2JG81 CDS 5' 203 decay incomplete TSL:2 Atp6v0a2- ENSMUST00000197087.4 3245 No - Retained intron - - TSL:1 202 protein Atp6v0a2- ENSMUST00000197931.1 663 No - Retained intron - - TSL:2 204 protein Atp6v0a2- ENSMUST00000200292.4 357 No - lncRNA - - TSL:3 207 protein Atp6v0a2- ENSMUST00000199526.1 126 No - lncRNA - - TSL:5 206 protein Page 6 of 8 https://www.alphaknockout.com 115.88 kb Forward strand 124.62Mb 124.64Mb 124.66Mb 124.68Mb 124.70Mb 124.72Mb Genes (Comprehensive set... Tctn2-201 >protein coding Atp6v0a2-203 >nonsense mediated decay Tctn2-207 >lncRNA Atp6v0a2-206 >lncRNA Tctn2-203 >lncRNA Atp6v0a2-202 >retained intron Atp6v0a2-207 >lncRNA Dnah10-201 >protein coding Atp6v0a2-201 >protein coding Atp6v0a2-205 >nonsense mediated decay Atp6v0a2-204 >retained intron Dnah10-203 >protein coding Contigs < AC242457.3 Genes < 4930404A12Rik-202lncRNA (Comprehensive set... < 4930404A12Rik-201lncRNA < Gm24854-201snRNA