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Mouse Dapp1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Dapp1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Dapp1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Dapp1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Dapp1 gene (NCBI Reference Sequence: NM_011932 ; Ensembl: ENSMUSG00000028159 ) is located on Mouse chromosome 3. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000029806). 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 Dapp1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-388F8 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: Inactivation of this gene invokes immune defects stemming from impaired B cell receptor crosslinking. Exon 3 starts from about 26.79% 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: 4305 bp, and the size of intron 3 for 3'-loxP site insertion: 11664 bp. The size of effective cKO region: ~634 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 3 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Dapp1 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7134bp) | A(28.67% 2045) | C(21.81% 1556) | T(28.17% 2010) | G(21.35% 1523) 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% chr3 - 137961826 137964825 3000 browser details YourSeq 126 365 535 3000 94.0% chr1 + 187513064 187513291 228 browser details YourSeq 118 387 542 3000 96.9% chr11 + 11554911 11555067 157 browser details YourSeq 97 405 535 3000 90.2% chr12 - 113453630 113453744 115 browser details YourSeq 84 405 590 3000 97.9% chr12 + 42519230 42519417 188 browser details YourSeq 80 405 489 3000 97.7% chr10 - 117404017 117404103 87 browser details YourSeq 72 429 505 3000 98.7% chr1 + 150129172 150129276 105 browser details YourSeq 65 468 534 3000 98.6% chr14 - 32777379 32777445 67 browser details YourSeq 49 366 475 3000 78.6% chr8 + 114498291 114498379 89 browser details YourSeq 47 393 446 3000 96.1% chr12 - 111024113 111024173 61 browser details YourSeq 47 1825 2177 3000 91.1% chr9 + 89966621 89967172 552 browser details YourSeq 44 1705 1800 3000 95.9% chr14 + 47014846 47014941 96 browser details YourSeq 43 422 494 3000 89.4% chr12 - 36711022 36711092 71 browser details YourSeq 42 1781 1853 3000 87.8% chr2 + 3630619 3630690 72 browser details YourSeq 41 367 422 3000 92.0% chr11 + 103843234 103843296 63 browser details YourSeq 38 694 771 3000 93.2% chr1 + 170687542 170687656 115 browser details YourSeq 37 1826 1953 3000 90.3% chr3 + 69668402 69668528 127 browser details YourSeq 35 407 442 3000 100.0% chr1 + 175488759 175488941 183 browser details YourSeq 34 267 310 3000 88.7% chr5 - 29261784 29261827 44 browser details YourSeq 34 1779 1840 3000 91.7% chr15 - 12727321 12727381 61 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% chr3 - 137958192 137961191 3000 browser details YourSeq 182 1983 2231 3000 87.3% chr2 - 156291570 156291796 227 browser details YourSeq 162 2058 2240 3000 95.1% chr12 + 111206223 111206413 191 browser details YourSeq 156 1957 2227 3000 83.0% chr4 + 130940574 130940835 262 browser details YourSeq 156 2049 2228 3000 93.4% chr12 + 76084326 76084505 180 browser details YourSeq 152 2054 2228 3000 94.2% chr9 - 99619282 99619460 179 browser details YourSeq 152 2054 2243 3000 90.9% chr7 - 127117341 127117531 191 browser details YourSeq 152 1996 2226 3000 85.2% chr14 + 76200097 76200289 193 browser details YourSeq 151 2054 2242 3000 89.4% chr8 - 22576286 22576469 184 browser details YourSeq 151 2055 2228 3000 93.7% chr11 - 119222606 119222780 175 browser details YourSeq 151 2055 2227 3000 93.7% chr11 - 86917936 86918108 173 browser details YourSeq 150 2059 2229 3000 94.2% chr1 + 128376383 128376741 359 browser details YourSeq 149 2054 2229 3000 93.6% chr19 - 7010665 7011232 568 browser details YourSeq 147 2055 2227 3000 93.1% chr13 - 67383641 67383815 175 browser details YourSeq 147 2063 2240 3000 91.6% chr3 + 19683755 19683933 179 browser details YourSeq 146 2059 2242 3000 87.8% chr6 - 83082232 83082410 179 browser details YourSeq 146 2055 2230 3000 92.1% chr5 + 29698166 29698344 179 browser details YourSeq 146 2056 2242 3000 90.6% chr4 + 141221580 141221767 188 browser details YourSeq 146 1814 2238 3000 80.6% chr4 + 42038633 42038845 213 browser details YourSeq 146 2057 2441 3000 84.4% chr11 + 83490615 83490897 283 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Dapp1 dual adaptor for phosphotyrosine and 3-phosphoinositides 1 [ Mus musculus (house mouse) ] Gene ID: 26377, updated on 10-Oct-2019 Gene summary Official Symbol Dapp1 provided by MGI Official Full Name dual adaptor for phosphotyrosine and 3-phosphoinositides 1 provided by MGI Primary source MGI:MGI:1347063 See related Ensembl:ENSMUSG00000028159 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 Bam32 Expression Ubiquitous expression in bladder adult (RPKM 5.5), liver E14 (RPKM 5.3) and 26 other tissues See more Orthologs human all Genomic context Location: 3; 3 G3 See Dapp1 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (137931007..137981549, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (137593970..137644513, complement) Chromosome 3 - NC_000069.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Dapp1 ENSMUSG00000028159 Description dual adaptor for phosphotyrosine and 3-phosphoinositides 1 [Source:MGI Symbol;Acc:MGI:1347063] Gene Synonyms Bam32 Location Chromosome 3: 137,931,007-137,981,545 reverse strand. GRCm38:CM000996.2 About this gene This gene has 7 transcripts (splice variants), 195 orthologues, 8 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 Dapp1- ENSMUST00000029806.12 3083 280aa ENSMUSP00000029806.6 Protein coding CCDS17864 Q3UK44 TSL:1 201 Q9QXT1 GENCODE basic APPRIS P1 Dapp1- ENSMUST00000136613.3 2950 239aa ENSMUSP00000119634.1 Protein coding CCDS80029 Q3TEK6 TSL:1 203 GENCODE basic Dapp1- ENSMUST00000199541.1 832 No - Retained - - TSL:NA 206 protein intron Dapp1- ENSMUST00000184222.5 962 No - lncRNA - - TSL:1 205 protein Dapp1- ENSMUST00000124827.7 782 No - lncRNA - - TSL:3 202 protein Dapp1- ENSMUST00000200596.1 439 No - lncRNA - - TSL:5 207 protein Dapp1- ENSMUST00000154716.1 429 No - lncRNA - - TSL:2 204 protein Page 6 of 8 https://www.alphaknockout.com 70.54 kb Forward strand Genes Lamtor3-203 >protein coding (Comprehensive set... Lamtor3-201 >protein coding Lamtor3-202 >lncRNA Lamtor3-205 >protein coding Lamtor3-207 >retained intron Lamtor3-206 >retained intron Lamtor3-208 >retained intron Contigs < AC146607.2 Genes < Dapp1-201protein coding (Comprehensive set... < Dapp1-203protein coding < Dapp1-205lncRNA < Dapp1-206retained intron < Dapp1-202lncRNA < Dapp1-204lncRNA < Dapp1-207lncRNA Regulatory Build Reverse strand 70.54 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 RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000029806 < Dapp1-201protein coding Reverse strand 50.54 kb ENSMUSP00000029... Superfamily SH2 domain superfamily SSF50729 SMART SH2 domain Pleckstrin homology domain Prints PR00678 SH2 domain Pfam SH2 domain Pleckstrin homology domain PROSITE profiles SH2 domain Pleckstrin homology domain PANTHER PTHR14336 PTHR14336:SF7 Gene3D SH2 domain superfamily PH-like domain superfamily CDD DAPP1, SH2 domain cd10573 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 240 280 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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