https://www.alphaknockout.com Mouse Ap3d1 Knockout Project (CRISPR/Cas9) Objective: To create a Ap3d1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ap3d1 gene (NCBI Reference Sequence: NM_007460 ; Ensembl: ENSMUSG00000020198 ) is located on Mouse chromosome 10. 31 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 31 (Transcript: ENSMUST00000020420). Exon 2~8 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: Mutant mice show coat and eye color dilution, platelet defects, lysosomal abnormalities, inner ear degeneration and neurological defects and model Hermansky-Pudlak storage pool deficiency syndrome. Exon 2 starts from about 2.7% of the coding region. Exon 2~8 covers 19.74% of the coding region. The size of effective KO region: ~9414 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 8 31 Legends Exon of mouse Ap3d1 Knockout region Page 2 of 9 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 Exon 2 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 570 bp section downstream of Exon 8 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 9 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(24.7% 494) | T(27.3% 546) | G(25.0% 500) Note: The 2000 bp section upstream of Exon 2 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(570bp) | A(17.72% 101) | C(24.39% 139) | T(32.28% 184) | G(25.61% 146) Note: The 570 bp section downstream of Exon 8 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 9 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% chr10 - 80732947 80734946 2000 browser details YourSeq 162 1022 1207 2000 94.7% chr4 - 155380434 155836588 456155 browser details YourSeq 160 1023 1250 2000 92.6% chr4 - 150456092 150456333 242 browser details YourSeq 156 724 1185 2000 83.8% chr2 - 154766326 154766573 248 browser details YourSeq 154 1022 1212 2000 88.2% chr19 + 28773051 28773236 186 browser details YourSeq 153 1022 1209 2000 91.0% chr4 + 127136807 127137001 195 browser details YourSeq 153 1022 1203 2000 93.3% chr10 + 56240337 56240523 187 browser details YourSeq 151 1024 1209 2000 92.7% chr15 - 100826546 100826735 190 browser details YourSeq 151 1015 1204 2000 87.8% chr19 + 28221071 28221249 179 browser details YourSeq 149 722 1175 2000 81.6% chr11 - 34984720 34984974 255 browser details YourSeq 148 1013 1187 2000 92.6% chr15 - 97771911 97772098 188 browser details YourSeq 147 1023 1209 2000 89.1% chr4 + 58816351 58816535 185 browser details YourSeq 147 1019 1207 2000 92.1% chr2 + 68847023 68847212 190 browser details YourSeq 146 1022 1207 2000 86.7% chr3 - 95158767 95158946 180 browser details YourSeq 146 1022 1209 2000 91.0% chr3 - 69772433 69772622 190 browser details YourSeq 146 1020 1205 2000 89.5% chr2 - 112711854 112712035 182 browser details YourSeq 146 1019 1203 2000 90.7% chr15 - 32775737 32775921 185 browser details YourSeq 146 1015 1206 2000 91.6% chr10 - 51673731 51673924 194 browser details YourSeq 146 1022 1205 2000 90.9% chr2 + 27437225 27437406 182 browser details YourSeq 146 1012 1209 2000 90.2% chr12 + 108676322 108676519 198 Note: The 2000 bp section upstream of Exon 2 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 570 1 570 570 100.0% chr10 - 80722963 80723532 570 browser details YourSeq 40 116 169 570 91.5% chr1 + 21138795 21138855 61 browser details YourSeq 39 97 154 570 76.2% chr10 + 62686762 62686804 43 browser details YourSeq 34 160 226 570 92.5% chr8 - 61706501 61706579 79 browser details YourSeq 34 157 225 570 90.5% chr6 - 95592004 95592490 487 browser details YourSeq 33 105 143 570 88.9% chr10 + 69312781 69312818 38 browser details YourSeq 32 152 209 570 92.2% chr1 + 188880860 188880926 67 browser details YourSeq 30 110 145 570 94.3% chr4 - 88439662 88439718 57 browser details YourSeq 28 205 234 570 96.7% chr7 - 119480375 119480404 30 browser details YourSeq 28 193 223 570 96.7% chr4 - 45250073 45250106 34 browser details YourSeq 28 155 182 570 100.0% chr17 - 87005683 87005710 28 browser details YourSeq 27 156 184 570 96.6% chr14 - 79447205 79447233 29 browser details YourSeq 27 200 238 570 79.0% chr1 - 60108897 60108934 38 browser details YourSeq 25 491 518 570 96.3% chr10 - 22843566 22843601 36 browser details YourSeq 25 155 179 570 100.0% chr14 + 121410791 121410815 25 browser details YourSeq 25 130 155 570 100.0% chr11 + 9215925 9215951 27 browser details YourSeq 25 134 176 570 79.1% chr1 + 131877962 131878004 43 browser details YourSeq 25 137 170 570 96.3% chr1 + 85672961 85672997 37 browser details YourSeq 24 152 175 570 100.0% chr6 - 40421929 40421952 24 browser details YourSeq 24 96 120 570 100.0% chr11 - 35721380 35721405 26 Note: The 570 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Ap3d1 adaptor-related protein complex 3, delta 1 subunit [ Mus musculus (house mouse) ] Gene ID: 11776, updated on 12-Aug-2019 Gene summary Official Symbol Ap3d1 provided by MGI Official Full Name adaptor-related protein complex 3, delta 1 subunit provided by MGI Primary source MGI:MGI:107734 See related Ensembl:ENSMUSG00000020198 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 mh; Ap3d; Bolvr; mocha; mBLVR1; AA407035 Expression Ubiquitous expression in testis adult (RPKM 54.8), ovary adult (RPKM 50.0) and 28 other tissues See more Orthologs human all Genomic context Location: 10 C1; 10 39.72 cM See Ap3d1 in Genome Data Viewer Exon count: 32 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (80706956..80742303, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (80169723..80204956, complement) Chromosome 10 - NC_000076.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Ap3d1 ENSMUSG00000020198 Description adaptor-related protein complex 3, delta 1 subunit [Source:MGI Symbol;Acc:MGI:107734] Gene Synonyms Bolvr, mBLVR1 Location Chromosome 10: 80,706,956-80,742,264 reverse strand. GRCm38:CM001003.2 About this gene This gene has 7 transcripts (splice variants), 213 orthologues, is a member of 1 Ensembl protein family and is associated with 38 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ap3d1- ENSMUST00000020420.8 4805 1199aa ENSMUSP00000020420.7 Protein coding CCDS35984 O54774 TSL:1 201 GENCODE basic APPRIS P1 Ap3d1- ENSMUST00000218610.1 786 116aa ENSMUSP00000151820.1 Nonsense mediated - A0A1W2P7Z2 CDS 5' 203 decay incomplete TSL:3 Ap3d1- ENSMUST00000219356.1 766 122aa ENSMUSP00000151355.1 Nonsense mediated - A0A1W2P6Q6 CDS 5' 205 decay incomplete TSL:3 Ap3d1- ENSMUST00000218125.1 3728 No - Retained intron - - TSL:1 202 protein Ap3d1- ENSMUST00000219816.1 858 No - Retained intron - - TSL:2 206 protein Ap3d1- ENSMUST00000219253.1 668 No - Retained intron - - TSL:2 204 protein Ap3d1- ENSMUST00000220183.1 422 No - Retained intron - - TSL:2 207 protein Page 7 of 9 https://www.alphaknockout.com 55.31 kb Forward strand 80.70Mb 80.71Mb 80.72Mb 80.73Mb 80.74Mb 80.75Mb Genes Izumo4-213 >retained intron (Comprehensive set... Izumo4-212 >retained intron Izumo4-202 >retained intron Izumo4-211 >retained intron Izumo4-205 >protein coding Izumo4-201 >protein coding Izumo4-209 >retained intron Izumo4-204 >retained intron Izumo4-203 >retained intron Izumo4-208 >retained intron Izumo4-206 >nonsense mediated decay Izumo4-207 >retained intron Izumo4-210 >retained intron Contigs AC152410.6 > Genes (Comprehensive set... < Mob3a-201protein coding < Ap3d1-201protein coding < Mob3a-202protein coding < Ap3d1-206retained intron < Ap3d1-207retained intron < Mob3a-203lncRNA < Ap3d1-203nonsense mediated decay < Ap3d1-205nonsense mediated decay < Ap3d1-202retained intron < Ap3d1-204retained intron Regulatory Build 80.70Mb 80.71Mb 80.72Mb 80.73Mb 80.74Mb 80.75Mb Reverse strand 55.31 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 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000020420 < Ap3d1-201protein coding Reverse strand 35.31 kb ENSMUSP00000020..