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Mouse Crhbp Conditional Knockout Project (CRISPR/Cas9)

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Mouse Crhbp Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Crhbp Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Crhbp conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Crhbp gene (NCBI Reference Sequence: NM_198408 ; Ensembl: ENSMUSG00000021680 ) is located on Mouse chromosome 13. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000221025). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Crhbp gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-49J13 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 disruptions in this gene display behavior indicative of increased anxiety. Male mice also show reduced food intake resulting in reduced growth between ages 7 and 15 weeks. Exon 4 starts from about 34.58% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 1458 bp, and the size of intron 4 for 3'-loxP site insertion: 1851 bp. The size of effective cKO region: ~711 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 2 3 4 5 7 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Crhbp 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. 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(7211bp) | A(26.54% 1914) | C(23.19% 1672) | T(27.65% 1994) | G(22.62% 1631) 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% chr13 - 95442529 95445528 3000 browser details YourSeq 42 2571 2616 3000 97.9% chr19 - 38197106 38197190 85 browser details YourSeq 40 2574 2618 3000 90.7% chr12 + 89268508 89268551 44 browser details YourSeq 34 1147 1224 3000 79.5% chr9 + 29313014 29313086 73 browser details YourSeq 31 2581 2612 3000 100.0% chr1 + 14330083 14330140 58 browser details YourSeq 29 2571 2612 3000 71.9% chr7 - 108595022 108595054 33 browser details YourSeq 27 418 456 3000 93.6% chr18 - 49592471 49592511 41 browser details YourSeq 27 2573 2612 3000 68.8% chr14 - 117412491 117412522 32 browser details YourSeq 27 405 455 3000 76.5% chr10 - 16093925 16093975 51 browser details YourSeq 23 422 446 3000 87.5% chr14 - 99911818 99911841 24 browser details YourSeq 22 1133 1157 3000 95.9% chr11 - 116544591 116544617 27 browser details YourSeq 21 1402 1422 3000 100.0% chr17 + 66049276 66049296 21 browser details YourSeq 21 1436 1456 3000 100.0% chr13 + 57313616 57313636 21 browser details YourSeq 21 1438 1458 3000 100.0% chr10 + 88274576 88274596 21 Note: The 3000 bp section upstream of Exon 4 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% chr13 - 95438818 95441817 3000 browser details YourSeq 117 771 921 3000 91.2% chr1 + 171432526 171432675 150 browser details YourSeq 114 771 918 3000 90.3% chr12 + 14632380 14632539 160 browser details YourSeq 112 760 934 3000 88.3% chr13 - 19637900 19638115 216 browser details YourSeq 108 772 921 3000 92.9% chr13 - 90922084 90922234 151 browser details YourSeq 107 768 921 3000 91.6% chr6 - 67296468 67296638 171 browser details YourSeq 105 779 921 3000 90.0% chr11 - 112087528 112087673 146 browser details YourSeq 105 776 905 3000 90.8% chr1 - 145716773 145716903 131 browser details YourSeq 105 789 918 3000 92.6% chr8 + 85313541 85313677 137 browser details YourSeq 104 763 904 3000 90.7% chr11 + 106477473 106477620 148 browser details YourSeq 103 777 908 3000 90.2% chr2 - 34701450 34701736 287 browser details YourSeq 103 772 908 3000 90.0% chr10 - 4396714 4396848 135 browser details YourSeq 103 771 904 3000 93.3% chr10 + 115413253 115413387 135 browser details YourSeq 103 793 921 3000 92.0% chr1 + 75295371 75295499 129 browser details YourSeq 101 774 908 3000 94.0% chr19 + 42499922 42500057 136 browser details YourSeq 101 769 912 3000 89.9% chr10 + 48553786 48553934 149 browser details YourSeq 99 769 905 3000 93.1% chr9 - 78493504 78493641 138 browser details YourSeq 99 772 905 3000 91.0% chr1 - 178271075 178271209 135 browser details YourSeq 99 793 911 3000 92.5% chrX + 137825933 137826252 320 browser details YourSeq 99 773 905 3000 92.3% chr12 + 75707463 75707596 134 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Crhbp corticotropin releasing hormone binding protein [ Mus musculus (house mouse) ] Gene ID: 12919, updated on 12-Aug-2019 Gene summary Official Symbol Crhbp provided by MGI Official Full Name corticotropin releasing hormone binding protein provided by MGI Primary source MGI:MGI:88497 See related Ensembl:ENSMUSG00000021680 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 CRH-BP Expression Biased expression in frontal lobe adult (RPKM 5.5), cortex adult (RPKM 5.5) and 7 other tissues See more Orthologs human all Genomic context Location: 13 D1; 13 49.5 cM See Crhbp in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (95431376..95444831, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (96201331..96214786, complement) Chromosome 13 - NC_000079.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Crhbp ENSMUSG00000021680 Description corticotropin releasing hormone binding protein [Source:MGI Symbol;Acc:MGI:88497] Gene Synonyms CRH-BP Location Chromosome 13: 95,431,371-95,444,924 reverse strand. GRCm38:CM001006.2 About this gene This gene has 2 transcripts (splice variants), 189 orthologues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Crhbp-202 ENSMUST00000221025.1 1779 322aa ENSMUSP00000152083.1 Protein coding CCDS26698 Q3UY07 Q60571 TSL:1 GENCODE basic APPRIS P1 Crhbp-201 ENSMUST00000045583.8 1701 329aa ENSMUSP00000042578.8 Protein coding - A0A217FL62 TSL:1 GENCODE basic 33.55 kb Forward strand 95.43Mb 95.44Mb 95.45Mb Contigs AC159263.3 > Genes (Comprehensive set... < Gm48712-201processed pseudogene < Crhbp-202protein coding < Crhbp-201protein coding Regulatory Build 95.43Mb 95.44Mb 95.45Mb Reverse strand 33.55 kb Regulation Legend Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000221025 < Crhbp-202protein coding Reverse strand 13.55 kb ENSMUSP00000152... Cleavage site (Sign... Superfamily Spermadhesin, CUB domain superfamily Pfam Corticotropin-releasing factor-binding protein PIRSF Corticotropin-releasing factor-binding protein PANTHER Corticotropin-releasing factor-binding protein All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 322 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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