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Mouse Crhbp Knockout Project (CRISPR/Cas9)

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Mouse Crhbp Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Crhbp Knockout Project (CRISPR/Cas9) Objective: To create a Crhbp 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 2~6 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: 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 2 starts from about 8.49% of the coding region. Exon 2~6 covers 75.57% of the coding region. The size of effective KO region: ~7865 bp. The KO 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 6 7 Legends Exon of mouse Crhbp Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 330 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 2000 bp section downstream of Exon 6 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 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(330bp) | A(18.18% 60) | C(34.85% 115) | T(29.39% 97) | G(17.58% 58) Note: The 330 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(2000bp) | A(28.15% 563) | C(20.45% 409) | T(31.0% 620) | G(20.4% 408) Note: The 2000 bp section downstream of Exon 6 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 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 330 1 330 330 100.0% chr13 - 95444270 95444599 330 browser details YourSeq 34 218 295 330 79.5% chr9 + 29313014 29313086 73 browser details YourSeq 23 43 68 330 96.2% chr19 - 26789206 26789239 34 browser details YourSeq 21 214 234 330 100.0% chr14 - 120791361 120791381 21 browser details YourSeq 21 230 251 330 100.0% chr15 + 101393549 101393571 23 browser details YourSeq 21 135 155 330 100.0% chr11 + 51553743 51553763 21 browser details YourSeq 20 202 221 330 100.0% chr18 - 81297580 81297599 20 browser details YourSeq 20 237 256 330 100.0% chr13 + 92911931 92911950 20 Note: The 330 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 2000 1 2000 2000 100.0% chr13 - 95434405 95436404 2000 browser details YourSeq 169 1296 1641 2000 88.7% chr1 - 23220216 23220533 318 browser details YourSeq 165 1294 1487 2000 91.2% chr12 - 101683525 101683704 180 browser details YourSeq 157 1289 1487 2000 89.9% chr18 + 19454477 19454663 187 browser details YourSeq 153 1305 1481 2000 95.9% chr11 + 102384498 102384992 495 browser details YourSeq 152 1291 1486 2000 88.5% chr18 + 57231766 57231951 186 browser details YourSeq 151 1307 1481 2000 93.7% chr15 - 99640356 99640530 175 browser details YourSeq 150 1290 1486 2000 92.5% chr10 + 80820226 80820421 196 browser details YourSeq 148 1325 1485 2000 96.3% chr1 - 84877261 84877423 163 browser details YourSeq 147 1296 1486 2000 88.9% chr16 + 61216730 61216917 188 browser details YourSeq 144 1308 1477 2000 93.5% chr6 - 21689132 21689304 173 browser details YourSeq 143 1324 1481 2000 95.6% chr12 + 12163135 12163298 164 browser details YourSeq 141 1334 1569 2000 93.9% chr7 + 81526885 81527371 487 browser details YourSeq 141 1326 1474 2000 98.0% chr5 + 136215052 136215204 153 browser details YourSeq 141 1334 1486 2000 96.1% chr10 + 11355906 11356058 153 browser details YourSeq 140 1326 1486 2000 93.8% chr3 - 130713581 130713743 163 browser details YourSeq 140 1292 1486 2000 87.5% chr11 - 6567506 6567669 164 browser details YourSeq 140 1307 1499 2000 93.8% chr6 + 147190501 147190844 344 browser details YourSeq 138 1294 1485 2000 86.6% chr10 - 107876334 107876502 169 browser details YourSeq 138 1320 1477 2000 94.4% chr17 + 23641294 23641473 180 Note: The 2000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 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 6 of 8 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 7 of 8 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 8 of 8.
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