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Mouse Cort Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cort conditional 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 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cort gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-46E8 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 increased circulating growth and adrenocorticotropin, decreased serum prolactin, and resistance and increased serum glucose level in male mice.

Exon 2 covers 63.3% of the coding region. Start codon is in exon 1, and stop codon is in exon 2. The size of intron 1 for 5'-loxP site insertion: 1053 bp. The size of effective cKO region: ~1190 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Cort cKO region loxP site

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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(6707bp) | A(23.1% 1549) | C(23.9% 1603) | T(26.93% 1806) | G(26.08% 1749)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr4 - 149125724 149128723 3000 browser details YourSeq 36 2915 2973 3000 95.0% chr2 + 173075581 173075639 59 browser details YourSeq 35 2889 2935 3000 90.5% chr5 - 130756806 130756857 52 browser details YourSeq 32 1098 1143 3000 92.2% chr11 - 98015399 98015447 49 browser details YourSeq 32 2899 2933 3000 87.9% chr17 + 3498976 3499008 33 browser details YourSeq 30 2890 2929 3000 77.2% chr19 - 4331525 4331560 36 browser details YourSeq 27 2889 2929 3000 83.9% chr6 + 119884045 119884083 39 browser details YourSeq 25 101 150 3000 76.0% chr2 + 38650116 38650167 52 browser details YourSeq 24 2917 2946 3000 96.2% chr7 - 51017436 51017466 31 browser details YourSeq 24 2903 2929 3000 84.0% chr18 + 33860401 33860425 25

Note: The 3000 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 3000 1 3000 3000 100.0% chr4 - 149122267 149125266 3000 browser details YourSeq 220 2546 2983 3000 89.3% chr13 + 55585441 55585826 386 browser details YourSeq 208 2549 2993 3000 98.7% chr1 + 72252685 72253268 584 browser details YourSeq 190 2530 2741 3000 97.1% chr7 + 73549203 73549432 230 browser details YourSeq 188 2546 2736 3000 99.5% chr13 - 64361837 64362049 213 browser details YourSeq 188 2547 2741 3000 98.5% chr18 + 56565683 56565878 196 browser details YourSeq 187 2546 2736 3000 99.0% chr8 + 106934843 106935033 191 browser details YourSeq 186 2546 2733 3000 99.5% chr11 + 116368743 116368930 188 browser details YourSeq 185 2547 2740 3000 98.0% chr5 - 135157807 135158003 197 browser details YourSeq 185 2502 2736 3000 96.6% chr12 - 111568113 111568348 236 browser details YourSeq 184 2547 2741 3000 97.5% chr11 - 72171330 72171526 197 browser details YourSeq 184 2530 2741 3000 93.3% chr3 + 27679151 27679361 211 browser details YourSeq 183 2547 2733 3000 97.9% chr14 - 47453891 47454076 186 browser details YourSeq 183 2547 2736 3000 96.9% chr11 - 53313134 53313321 188 browser details YourSeq 183 2530 2741 3000 95.6% chr10 - 4351204 4351422 219 browser details YourSeq 183 2547 2732 3000 99.5% chr10 + 34221790 34222000 211 browser details YourSeq 182 2547 2740 3000 97.5% chr19 - 37152997 37153194 198 browser details YourSeq 181 2531 2736 3000 94.7% chr2 - 57295086 57295311 226 browser details YourSeq 181 2528 2741 3000 93.1% chr10 - 34305898 34306108 211 browser details YourSeq 181 2539 2734 3000 96.9% chr5 + 65313941 65314151 211

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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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 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 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

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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

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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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

PTHR22980:SF2

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.

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