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Mouse Snap23 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Snap23 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Snap23 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Snap23 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Snap23 gene (NCBI Reference Sequence: NM_001177792 ; Ensembl: ENSMUSG00000027287 ) is located on Mouse chromosome 2. 9 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000116437). Exon 3~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Snap23 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-117D14 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: Mice homozygous for a knock-out allele exhibit lethality prior to E3.5. Exon 3 starts from about 8.75% of the coding region. The knockout of Exon 3~5 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 785 bp, and the size of intron 5 for 3'-loxP site insertion: 4386 bp. The size of effective cKO region: ~1626 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 5' gRNA region gRNA region 3' 1 2 3 4 5 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Snap23 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(8126bp) | A(28.13% 2286) | C(18.8% 1528) | T(31.05% 2523) | G(22.02% 1789) 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% chr2 + 120581902 120584901 3000 browser details YourSeq 311 993 2877 3000 93.6% chr7 - 116109801 116200422 90622 browser details YourSeq 208 880 1145 3000 93.4% chr2 + 29545755 29546023 269 browser details YourSeq 200 880 1147 3000 95.1% chr8 - 111672052 112152308 480257 browser details YourSeq 198 860 1146 3000 90.6% chr2 + 180767204 180767492 289 browser details YourSeq 197 897 1145 3000 92.3% chr10 + 22059726 22486515 426790 browser details YourSeq 192 880 1145 3000 91.0% chr2 - 34846601 34846859 259 browser details YourSeq 184 827 1140 3000 91.5% chr13 + 63781799 63782405 607 browser details YourSeq 183 830 1145 3000 92.3% chr9 + 120630923 120631243 321 browser details YourSeq 175 746 1145 3000 80.8% chr1 + 164517441 164517848 408 browser details YourSeq 164 959 1145 3000 94.5% chr13 - 22032418 22032603 186 browser details YourSeq 158 2698 2895 3000 90.5% chr10 - 76452171 76452547 377 browser details YourSeq 158 959 1184 3000 90.7% chrX + 144251118 144251375 258 browser details YourSeq 156 2700 2890 3000 91.8% chr11 - 95886162 95886351 190 browser details YourSeq 154 2700 2885 3000 92.8% chr11 - 120653119 120653524 406 browser details YourSeq 154 2704 2890 3000 91.9% chr5 + 66020087 66020275 189 browser details YourSeq 154 2718 2890 3000 94.8% chr11 + 85634220 85634393 174 browser details YourSeq 153 887 1125 3000 86.0% chr1 - 133189319 133189546 228 browser details YourSeq 153 2699 2885 3000 91.1% chr17 + 87347929 87348113 185 browser details YourSeq 153 702 1098 3000 90.6% chr1 + 136566555 136567115 561 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% chr2 + 120586528 120589527 3000 browser details YourSeq 317 809 1231 3000 90.8% chr4 - 150697748 150993826 296079 browser details YourSeq 291 798 1197 3000 89.5% chr17 + 15613763 15614198 436 browser details YourSeq 198 759 1007 3000 90.0% chr2 + 172784358 172784607 250 browser details YourSeq 197 746 1011 3000 91.3% chr17 + 6647013 6647361 349 browser details YourSeq 191 800 1433 3000 83.0% chr6 - 53886857 53887098 242 browser details YourSeq 191 762 1054 3000 87.8% chr15 - 61964902 61965196 295 browser details YourSeq 190 798 1034 3000 90.3% chr4 + 150081510 150081748 239 browser details YourSeq 190 764 1034 3000 86.9% chr15 + 27134180 27134456 277 browser details YourSeq 190 798 1034 3000 92.1% chr10 + 110758898 110759157 260 browser details YourSeq 189 799 1039 3000 91.0% chr4 - 152209222 152209464 243 browser details YourSeq 187 766 1031 3000 86.8% chr8 - 9038700 9038968 269 browser details YourSeq 187 771 1011 3000 88.8% chr8 + 47969022 47969262 241 browser details YourSeq 187 798 1039 3000 90.6% chr6 + 100701947 100702202 256 browser details YourSeq 187 798 1152 3000 86.8% chr13 + 51781428 51781752 325 browser details YourSeq 186 798 1039 3000 89.8% chr9 - 118459553 118459796 244 browser details YourSeq 186 798 1039 3000 91.2% chr14 + 64878432 64878676 245 browser details YourSeq 185 798 1039 3000 90.1% chr5 - 75378345 75378588 244 browser details YourSeq 184 798 1433 3000 82.2% chr6 + 4769733 4769996 264 browser details YourSeq 184 798 1033 3000 90.8% chr15 + 31264024 31264261 238 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: Snap23 synaptosomal-associated protein 23 [ Mus musculus (house mouse) ] Gene ID: 20619, updated on 28-Sep-2019 Gene summary Official Symbol Snap23 provided by MGI Official Full Name synaptosomal-associated protein 23 provided by MGI Primary source MGI:MGI:109356 See related Ensembl:ENSMUSG00000027287 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 Sndt; 23kDa; Syndet; SNAP-23; AA408749 Expression Broad expression in placenta adult (RPKM 10.3), bladder adult (RPKM 8.3) and 24 other tissues See more Orthologs human all Genomic context Location: 2 60.37 cM; 2 E5 See Snap23 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (120567655..120601255) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (120393407..120426458) Chromosome 2 - NC_000068.7 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Snap23 ENSMUSG00000027287 Description synaptosomal-associated protein 23 [Source:MGI Symbol;Acc:MGI:109356] Gene Synonyms SNAP-23, Sndt, Syndet Location Chromosome 2: 120,567,671-120,601,255 forward strand. GRCm38:CM000995.2 About this gene This gene has 9 transcripts (splice variants), 213 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Snap23- ENSMUST00000116437.7 2730 221aa ENSMUSP00000112138.1 Protein coding CCDS50681 Q9D3L3 TSL:1 203 GENCODE basic Snap23- ENSMUST00000110711.8 2723 210aa ENSMUSP00000106339.2 Protein coding CCDS16622 A2AKH4 TSL:1 202 O09044 GENCODE basic APPRIS P1 Snap23- ENSMUST00000028743.9 2658 210aa ENSMUSP00000028743.3 Protein coding CCDS16622 A2AKH4 TSL:1 201 O09044 GENCODE basic APPRIS P1 Snap23- ENSMUST00000142278.1 751 161aa ENSMUSP00000116935.1 Protein coding - B0R030 CDS 3' 206 incomplete TSL:3 Snap23- ENSMUST00000153580.7 481 85aa ENSMUSP00000121509.1 Protein coding - B0R029 CDS 3' 209 incomplete TSL:2 Snap23- ENSMUST00000150611.7 791 98aa ENSMUSP00000119652.1 Nonsense mediated - E9Q8A1 TSL:5 208 decay Snap23- ENSMUST00000147864.7 791 No - Retained intron - - TSL:2 207 protein Snap23- ENSMUST00000137144.1 753 No - Retained intron - - TSL:3 205 protein Snap23- ENSMUST00000130759.1 405 No - lncRNA - - TSL:5 204 protein Page 6 of 8 https://www.alphaknockout.com 53.59 kb Forward strand 120.56Mb 120.57Mb 120.58Mb 120.59Mb 120.60Mb 120.61Mb Genes (Comprehensive set... Snap23-202 >protein coding Haus2-203 >protein coding Snap23-201 >protein coding Haus2-202 >protein coding Snap23-203 >protein coding Snap23-208 >nonsense mediated decay Haus2-201 >nonsense mediated decay Snap23-209 >protein coding Snap23-204 >lncRNA Snap23-207 >retained intron Snap23-205 >retained intron Snap23-206 >protein coding Contigs AL772299.10 > Genes < Zfp106-201protein coding < Lrrc57-207retained intron (Comprehensive set... < Zfp106-205retained intron < Lrrc57-204protein coding < Zfp106-203protein coding < Lrrc57-203protein coding < Zfp106-206lncRNA < Lrrc57-202protein coding < Lrrc57-201protein coding
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