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Mouse Cort Knockout Project (CRISPR/Cas9)

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Mouse Cort Knockout Project (CRISPR/Cas9) http://beta.alphaknockout.cyagen.net Mouse Cort Knockout Project (CRISPR/Cas9) Objective: To create a Cort knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cort gene ( NCBI Reference Sequence: NM_007745 ; Ensembl: ENSMUSG00000028971 ) is located on mouse chromosome 4. 2 exons are identified , with the ATG start codon in exon 1 and the TAA stop codon in exon 2 (Transcript: ENSMUST00000030815). Exon 1~2 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 a knock-out allele exhibit increased circulating growth hormone and adrenocorticotropin, decreased serum prolactin, and insulin resistance and increased serum glucose level in male mice. Exon 1 starts from the coding region. Exon 1~2 covers 100.0% of the coding region. The size of effective KO region: ~1731 bp. The expression of mouse Cenps may be affected by deletion of this KO region. Page 1 of 8 http://beta.alphaknockout.cyagen.net Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 Legends Knockout region Exon of mouse Cort Page 2 of 8 http://beta.alphaknockout.cyagen.net Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 1 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. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream 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. Page 3 of 8 http://beta.alphaknockout.cyagen.net Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(19.8% 396) | C(24.3% 486) | G(29.15% 583) | T(26.75% 535) Note: The 2000 bp section upstream of Exon 1 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(23.5% 470) | C(23.55% 471) | G(24.65% 493) | T(28.3% 566) Note: The 2000 bp section downstream 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. Page 4 of 8 http://beta.alphaknockout.cyagen.net BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr4 - 149126764 149128763 2000 browser details YourSeq 32 1138 1183 2000 92.2% chr11 - 98015399 98015447 49 browser details YourSeq 23 645 671 2000 84.7% chr1 + 56250092 56250117 26 browser details YourSeq 22 1422 1443 2000 100.0% chr8 - 106550195 106550216 22 browser details YourSeq 22 1153 1174 2000 100.0% chr13 + 11918655 11918676 22 browser details YourSeq 21 1293 1313 2000 100.0% chr1 - 49536131 49536151 21 browser details YourSeq 20 1267 1288 2000 95.5% chr1 + 9776780 9776801 22 Note: The 2000 bp section upstream of Exon 1 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% chr4 - 149123034 149125033 2000 browser details YourSeq 49 271 360 2000 91.7% chr6 - 120640645 120640774 130 browser details YourSeq 37 1301 1398 2000 89.4% chr15 - 103440726 103440967 242 browser details YourSeq 37 161 316 2000 87.5% chr7 + 16748316 16748469 154 browser details YourSeq 36 79 304 2000 55.0% chr7 + 3235494 3235537 44 browser details YourSeq 31 310 353 2000 91.9% chr16 - 37603101 37603146 46 browser details YourSeq 30 129 191 2000 96.9% chr7 + 143029218 143029281 64 browser details YourSeq 29 1297 1355 2000 74.6% chr2 - 168693589 168693647 59 browser details YourSeq 29 1298 1348 2000 96.8% chr17 - 83665914 83665965 52 browser details YourSeq 28 179 215 2000 96.9% chr15 + 82310092 82310134 43 browser details YourSeq 27 322 354 2000 93.6% chr13 + 44830189 44830223 35 browser details YourSeq 26 1385 1424 2000 82.5% chr1 - 181205262 181205301 40 browser details YourSeq 25 179 204 2000 100.0% chr9 - 108926265 108926292 28 browser details YourSeq 25 1304 1348 2000 72.8% chr15 - 73230360 73230403 44 browser details YourSeq 25 83 110 2000 96.5% chr13 + 108445325 108445356 32 browser details YourSeq 24 1293 1318 2000 96.2% chr1 + 85249046 85249071 26 browser details YourSeq 24 125 148 2000 100.0% chr1 + 67975448 67975471 24 browser details YourSeq 23 1298 1348 2000 72.6% chr10 - 70174361 70174411 51 browser details YourSeq 21 1298 1318 2000 100.0% chr16 + 12202778 12202798 21 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 http://beta.alphaknockout.cyagen.net Gene and protein information: Cort cortistatin [ Mus musculus (house mouse) ] Gene ID: 12854, updated on 12-Aug-2019 Gene summary Official Symbol Cort provided by MGI Official Full Name cortistatin provided by MGI Primary source MGI:MGI:109538 See related Ensembl:ENSMUSG00000028971 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as CST; PCST Summary This gene encodes a member of the somatostatin family of multifunctional peptides attributed with neurohormone, Expression neurotransmitter/modulator and autocrine/paracrine actions. The encoded preproprotein undergoes proteolytic processing to generate a mature functional peptide that can bind to somatostatin receptors. Mice lacking the encoded protein exhibit elevated levels of growth hormone in the plasma without major changes in somatic growth and have exacerbated nociceptive responses to neuropathic and inflammatory pain sensitization. Transgenic mice overexpressing the encoded protein in neurons do not express long-term potentiation in the dentate gyrus and exhibit deficits in synaptic plasticity and learning. [provided by RefSeq, Nov 2015] Orthologs Biased expression in frontal lobe adult (RPKM 2.9), cortex adult (RPKM 2.4) and 12 other tissues See more human all Genomic context Location: 4 E2; 4 78.89 cM See Cort in Genome Data Viewer Exon count: 2 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (149125034..149126763, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (148499300..148500850, complement) Chromosome 4 - NC_000070.6 Page 6 of 8 http://beta.alphaknockout.cyagen.net Transcript information: This gene has 1 transcript Gene: Cort ENSMUSG00000028971 Description cortistatin [Source:MGI Symbol;Acc:MGI:109538] Gene Synonyms CST, PCST Location Chromosome 4: 149,125,034-149,126,763 reverse strand. GRCm38:CM000997.2 About this gene This gene has 1 transcript (splice variant), 61 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cort-201 ENSMUST00000030815.2 677 109aa ENSMUSP00000030815.2 Protein coding CCDS18953 P56469 TSL:1 GENCODE basic APPRIS P1 21.73 kb Forward strand 149.120Mb 149.125Mb 149.130Mb 149.135Mb Genes Dffa-201 >protein coding (Comprehensive set... Dffa-202 >protein coding Contigs AL611967.21 > AL731655.8 > Genes (Comprehensive set... < Cort-201protein coding < Cenps-201protein coding < Cenps-203nonsense mediated decay < Cenps-207protein coding < Cenps-204retained intron < Cenps-206protein coding < Cenps-205protein coding < Cenps-202protein coding Regulatory Build 149.120Mb 149.125Mb 149.130Mb 149.135Mb Reverse strand 21.73 kb Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Page 7 of 8 http://beta.alphaknockout.cyagen.net Transcript: ENSMUST00000030815 < Cort-201protein coding Reverse strand 1.73 kb ENSMUSP00000030... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Pfam Somatostatin/Cortistatin, C-terminal PIRSF Somatostatin PANTHER PTHR22980:SF2 PTHR22980 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 10 20 30 40 50 60 70 80 90 109 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC, VectorBuilder. Page 8 of 8.
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