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Mouse Ap2a2 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ap2a2 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ap2a2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ap2a2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ap2a2 gene (NCBI Reference Sequence: NM_007459 ; Ensembl: ENSMUSG00000002957 ) is located on Mouse chromosome 7. 22 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 22 (Transcript: ENSMUST00000003038). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ap2a2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-271D24 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: Exon 3 starts from about 4.87% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 9118 bp, and the size of intron 3 for 3'-loxP site insertion: 1086 bp. The size of effective cKO region: ~643 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 3 4 22 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ap2a2 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. 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(7143bp) | A(23.14% 1653) | C(22.36% 1597) | T(29.16% 2083) | G(25.34% 1810) 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 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 + 141594224 141597223 3000 browser details YourSeq 153 1 554 3000 87.0% chr2 + 34402256 34402701 446 browser details YourSeq 145 1 247 3000 87.9% chr18 + 33593241 33593401 161 browser details YourSeq 145 1 167 3000 96.2% chr10 + 81708281 81708513 233 browser details YourSeq 144 1 155 3000 96.8% chr18 - 5531288 5531456 169 browser details YourSeq 144 1 162 3000 96.2% chr3 + 95548606 95548773 168 browser details YourSeq 143 1 154 3000 96.8% chr18 - 19494788 19494949 162 browser details YourSeq 143 1 154 3000 96.8% chr11 - 45366082 45366236 155 browser details YourSeq 142 1 157 3000 96.2% chr13 - 11188703 11188861 159 browser details YourSeq 142 1 156 3000 94.2% chr1 - 124612860 124613014 155 browser details YourSeq 142 1 150 3000 97.4% chr18 + 82435932 82436081 150 browser details YourSeq 142 1 152 3000 96.8% chr14 + 48444603 48444754 152 browser details YourSeq 141 1 151 3000 96.7% chr17 - 41171107 41171257 151 browser details YourSeq 141 1 149 3000 97.4% chr14 - 70424280 70424428 149 browser details YourSeq 141 1 147 3000 98.0% chr13 - 23671287 23671433 147 browser details YourSeq 141 1 153 3000 96.1% chr12 - 24816136 24816288 153 browser details YourSeq 141 1 153 3000 96.1% chr11 - 40946031 40946183 153 browser details YourSeq 141 1 153 3000 96.1% chr1 - 35262489 35262641 153 browser details YourSeq 141 1 149 3000 97.4% chr2 + 171485488 171485636 149 browser details YourSeq 141 1 150 3000 97.4% chr2 + 123753645 123753798 154 Note: The 3000 bp section upstream of Exon 3 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 + 141597867 141600866 3000 browser details YourSeq 168 1293 1491 3000 96.7% chrX + 151213738 151214652 915 browser details YourSeq 166 1276 1461 3000 93.6% chr1 - 38654725 38654909 185 browser details YourSeq 166 1270 1461 3000 92.6% chr16 + 20503800 20503989 190 browser details YourSeq 163 1293 1606 3000 93.6% chr11 - 57796350 57796830 481 browser details YourSeq 163 1273 1461 3000 93.7% chr9 + 78504052 78504246 195 browser details YourSeq 163 1270 1461 3000 93.0% chr2 + 25004744 25004934 191 browser details YourSeq 163 1283 1461 3000 96.1% chr18 + 35304827 35305022 196 browser details YourSeq 162 1272 1458 3000 91.9% chr3 + 152140342 152140526 185 browser details YourSeq 161 1277 1459 3000 95.6% chr7 + 27283979 27284172 194 browser details YourSeq 160 1283 1469 3000 93.1% chr5 - 25214367 25214577 211 browser details YourSeq 160 1283 1461 3000 95.6% chr4 - 40796524 40796710 187 browser details YourSeq 160 1271 1459 3000 94.5% chr2 - 157275836 157276035 200 browser details YourSeq 158 1279 1462 3000 94.4% chr5 - 142491211 142491400 190 browser details YourSeq 157 1273 1461 3000 92.5% chr7 - 47017585 47017780 196 browser details YourSeq 157 1274 1461 3000 92.5% chr5 - 135201209 135201548 340 browser details YourSeq 157 1283 1461 3000 93.9% chr5 - 32710823 32711001 179 browser details YourSeq 157 1270 1462 3000 91.1% chr4 - 127018960 127019160 201 browser details YourSeq 157 1273 1462 3000 89.8% chr15 + 89564043 89564219 177 browser details YourSeq 156 1294 1461 3000 96.5% chr3 - 82985702 82985869 168 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ap2a2 adaptor-related protein complex 2, alpha 2 subunit [ Mus musculus (house mouse) ] Gene ID: 11772, updated on 16-Sep-2019 Gene summary Official Symbol Ap2a2 provided by MGI Official Full Name adaptor-related protein complex 2, alpha 2 subunit provided by MGI Primary source MGI:MGI:101920 See related Ensembl:ENSMUSG00000002957 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 L25; Adtab; C78001; AF006990; AW146353; 2410074K14Rik Expression Ubiquitous expression in kidney adult (RPKM 33.9), duodenum adult (RPKM 33.3) and 28 other tissues See more Orthologs human all Genomic context Location: 7; 7 F5 See Ap2a2 in Genome Data Viewer Exon count: 23 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (141562180..141633011) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (148748079..148818910) Chromosome 7 - NC_000073.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Ap2a2 ENSMUSG00000002957 Description adaptor-related protein complex 2, alpha 2 subunit [Source:MGI Symbol;Acc:MGI:101920] Gene Synonyms 2410074K14Rik, Adtab, L25, alpha-C adaptin, alpha-adaptin C Location Chromosome 7: 141,562,173-141,633,011 forward strand. GRCm38:CM001000.2 About this gene This gene has 3 transcripts (splice variants), 141 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ap2a2-201 ENSMUST00000003038.11 4646 938aa ENSMUSP00000003038.8 Protein coding CCDS40188 P17427 TSL:1 GENCODE basic APPRIS P1 Ap2a2-202 ENSMUST00000201261.1 590 131aa ENSMUSP00000144090.1 Protein coding - A0A0J9YUA7 CDS 3' incomplete TSL:3 Ap2a2-203 ENSMUST00000201320.1 1549 No protein - Retained intron - - TSL:1 90.84 kb Forward strand 141.56Mb 141.58Mb 141.60Mb 141.62Mb 141.64Mb Genes (Comprehensive set... Ap2a2-201 >protein coding Ap2a2-203 >retained intron Mir7063-201 >miRNA Ap2a2-202 >protein coding Contigs AC158224.6 > AC102524.11 > Genes < Gm16982-201lncRNA < Muc6-202protein coding (Comprehensive set... < Muc6-201protein coding < Muc6-203protein coding Regulatory Build 141.56Mb 141.58Mb 141.60Mb 141.62Mb 141.64Mb Reverse strand 90.84 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000003038 70.84 kb Forward strand Ap2a2-201 >protein coding ENSMUSP00000003... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Armadillo-type fold Coatomer/calthrin adaptor appendage, C-terminal subdomain Clathrin adaptor, appendage, Ig-like subdomain superfamily SMART Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain Pfam Clathrin/coatomer adaptor, adaptin-like, N-terminal Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain PIRSF Adaptor protein complex AP-2, alpha subunit PANTHER PTHR22780 PTHR22780:SF30 Gene3D Armadillo-like helical 2.60.40.1230 TBP domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 800 938 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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