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Mouse Sptbn5 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Sptbn5 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Sptbn5 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Sptbn5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Sptbn5 gene (NCBI Reference Sequence: NM_001370938 ; Ensembl: ENSMUSG00000074899 ) is located on Mouse chromosome 2. 66 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 66 (Transcript: ENSMUST00000156159). Exon 15~17 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Sptbn5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-250P21 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 15 starts from about 25.37% of the coding region. The knockout of Exon 15~17 will result in frameshift of the gene. The size of intron 14 for 5'-loxP site insertion: 1113 bp, and the size of intron 17 for 3'-loxP site insertion: 923 bp. The size of effective cKO region: ~1526 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 5' gRNA region gRNA region 3' 1 13 14 15 16 17 18 19 66 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Sptbn5 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(8026bp) | A(24.33% 1953) | C(25.74% 2066) | T(22.55% 1810) | G(27.37% 2197) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. 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% chr2 - 120072774 120075773 3000 browser details YourSeq 125 1 164 3000 95.0% chr4 + 155766046 155766419 374 browser details YourSeq 120 1 128 3000 96.9% chrX + 162219666 162219793 128 browser details YourSeq 114 2411 2582 3000 87.0% chr9 - 25134626 25134824 199 browser details YourSeq 114 2416 2567 3000 89.6% chr1 + 66952963 66953143 181 browser details YourSeq 110 1 120 3000 95.9% chr14 + 27225721 27225840 120 browser details YourSeq 104 2420 2574 3000 90.3% chr12 - 76783487 76783667 181 browser details YourSeq 104 2427 2575 3000 87.4% chr10 - 12396663 12396829 167 browser details YourSeq 102 2414 2549 3000 88.8% chr12 - 86333235 86333369 135 browser details YourSeq 101 2413 2574 3000 91.2% chr9 + 49846713 49846902 190 browser details YourSeq 98 2411 2549 3000 87.2% chr4 - 127252890 127253028 139 browser details YourSeq 96 2426 2584 3000 88.0% chr5 + 86269450 86270065 616 browser details YourSeq 95 2418 2574 3000 89.4% chr10 - 18039410 18039583 174 browser details YourSeq 95 2419 2549 3000 88.0% chrX + 77733964 77734096 133 browser details YourSeq 95 2425 2562 3000 89.3% chr17 + 48764658 48765241 584 browser details YourSeq 95 2425 2567 3000 88.0% chr1 + 42916566 42916719 154 browser details YourSeq 94 2421 2567 3000 88.0% chr9 + 54993542 54993708 167 browser details YourSeq 93 2424 2573 3000 91.8% chrX + 151972613 151972980 368 browser details YourSeq 92 2416 2552 3000 84.9% chr8 + 105206141 105206278 138 browser details YourSeq 92 2415 2549 3000 87.7% chr14 + 72788088 72788220 133 Note: The 3000 bp section upstream of Exon 15 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 - 120068248 120071247 3000 browser details YourSeq 113 262 427 3000 87.6% chr4 + 106001926 106002096 171 browser details YourSeq 107 283 457 3000 81.3% chr18 - 62499344 62499517 174 browser details YourSeq 89 272 537 3000 73.4% chr5 + 136960712 136960988 277 browser details YourSeq 84 118 533 3000 82.4% chr2 - 130054990 130055435 446 browser details YourSeq 70 303 529 3000 74.4% chr17 - 7581460 7581701 242 browser details YourSeq 70 251 446 3000 87.3% chr4 + 57907459 57907659 201 browser details YourSeq 66 307 395 3000 86.5% chr6 - 107064080 107064165 86 browser details YourSeq 63 291 380 3000 85.6% chr5 - 72617599 72617689 91 browser details YourSeq 63 287 374 3000 86.4% chr5 + 67075207 67075295 89 browser details YourSeq 44 244 538 3000 82.4% chr17 - 13338438 13338792 355 browser details YourSeq 41 361 417 3000 86.0% chr3 + 54599746 54599802 57 browser details YourSeq 36 389 434 3000 89.2% chr14 + 62307638 62307683 46 browser details YourSeq 35 581 654 3000 81.4% chr14 + 5987866 5987936 71 browser details YourSeq 35 581 654 3000 81.4% chr14 + 5645831 5645901 71 browser details YourSeq 32 2180 2241 3000 94.6% chr5 + 16848946 16849012 67 browser details YourSeq 31 402 446 3000 84.5% chr8 - 66851337 66851381 45 browser details YourSeq 31 304 345 3000 87.5% chr3 + 137467447 137467488 42 browser details YourSeq 29 361 397 3000 89.2% chr1 + 89265927 89265963 37 browser details YourSeq 27 391 430 3000 93.8% chr18 - 19597526 19597566 41 Note: The 3000 bp section downstream of Exon 17 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Sptbn5 spectrin beta, non-erythrocytic 5 [ Mus musculus (house mouse) ] Gene ID: 640524, updated on 10-Oct-2019 Gene summary Official Symbol Sptbn5 provided by MGI Official Full Name spectrin beta, non-erythrocytic 5 provided by MGI Primary source MGI:MGI:2685200 See related Ensembl:ENSMUSG00000074899 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 Gm354; Spnb5; EG640524 Expression Low expression observed in reference dataset See more Orthologs human all Genomic context Location: 2; 2 E5 See Sptbn5 in Genome Data Viewer Exon count: 68 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (120041493..120088913, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (119867830..119911414, complement) Chromosome 2 - NC_000068.7 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Sptbn5 ENSMUSG00000074899 Description spectrin beta, non-erythrocytic 5 [Source:MGI Symbol;Acc:MGI:2685200] Gene Synonyms EG640524, Spnb5 Location Chromosome 2: 120,041,493-120,085,678 reverse strand. GRCm38:CM000995.2 About this gene This gene has 1 transcript (splice variant), 140 orthologues, 36 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS Flags Sptbn5-201 ENSMUST00000156159.3 11654 3624aa ENSMUSP00000158705.1 Protein coding - TSL:5 GENCODE basic APPRIS P1 64.19 kb Forward strand 120.04Mb 120.05Mb 120.06Mb 120.07Mb 120.08Mb 120.09Mb Genes Gm28042-204 >nonsense mediated decay (Comprehensive set... Gm28042-202 >protein coding Gm28042-203 >protein coding Jmjd7-201 >protein codingPla2g4b-202 >retained intron Jmjd7-203 >processed transcript Gm28042-201 >nonsense mediated decay Pla2g4b-203 >retained intron Pla2g4b-205 >retained intron Pla2g4b-201 >protein coding Pla2g4b-204 >retained intron Pla2g4b-206 >retained intron Contigs < AL833774.4 Genes < Sptbn5-201protein coding < Ehd4-201protein coding (Comprehensive set... Regulatory Build 120.04Mb 120.05Mb 120.06Mb 120.07Mb 120.08Mb 120.09Mb Reverse strand 64.19 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000156159 < Sptbn5-201protein coding Reverse strand 44.19 kb ENSMUSP00000158... Low complexity (Seg) All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 400 800 1200 1600 2000 2400 2800 3200 3624 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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