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Mouse Cutc Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cutc conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cutc gene (NCBI Reference Sequence: NM_001113562 ; Ensembl: ENSMUSG00000025193 ) is located on Mouse chromosome 19. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000112047). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cutc gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-63D21 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 5 starts from about 49.14% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 629 bp, and the size of intron 6 for 3'-loxP site insertion: 1953 bp. The size of effective cKO region: ~2818 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 3 4 5 6 7 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cutc Homology arm 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. 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(9318bp) | A(27.98% 2607) | C(20.21% 1883) | T(31.69% 2953) | G(20.12% 1875)

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% chr19 + 43757440 43760439 3000 browser details YourSeq 568 8 930 3000 86.8% chrX - 150759692 150760586 895 browser details YourSeq 565 1 929 3000 88.5% chr7 + 97908609 97909524 916 browser details YourSeq 553 1 929 3000 88.5% chr13 - 106781027 106781944 918 browser details YourSeq 551 1 879 3000 88.2% chr19 + 23685293 23686155 863 browser details YourSeq 548 31 1080 3000 86.8% chr2 + 91608258 91609627 1370 browser details YourSeq 546 31 928 3000 88.9% chr2 + 155455985 155456860 876 browser details YourSeq 545 2 923 3000 86.6% chr16 - 16005980 16006871 892 browser details YourSeq 543 1 902 3000 87.3% chr10 - 109908842 109909726 885 browser details YourSeq 522 2 898 3000 88.0% chr19 - 30369964 30370849 886 browser details YourSeq 520 21 904 3000 89.6% chr2 + 144879947 144880812 866 browser details YourSeq 512 1 929 3000 87.1% chr2 - 24236260 24237164 905 browser details YourSeq 497 19 917 3000 89.1% chr7 + 108958696 108959571 876 browser details YourSeq 497 1 898 3000 89.3% chr6 + 65844408 65845312 905 browser details YourSeq 489 62 929 3000 87.4% chr3 + 67649853 67650657 805 browser details YourSeq 487 1 929 3000 91.6% chr8 - 31365432 31366362 931 browser details YourSeq 487 60 929 3000 83.2% chr4 + 17018749 17019588 840 browser details YourSeq 486 1 930 3000 87.6% chr16 - 87064652 87065566 915 browser details YourSeq 485 20 902 3000 85.9% chr11 + 92838285 92839145 861 browser details YourSeq 484 173 929 3000 90.1% chr10 - 11270978 11271748 771

Note: The 3000 bp section upstream of Exon 5 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% chr19 + 43763258 43766257 3000 browser details YourSeq 116 1324 1487 3000 83.3% chr1 - 191545386 191545536 151 browser details YourSeq 113 1304 1474 3000 83.5% chr15 - 23941843 23942000 158 browser details YourSeq 111 1303 1479 3000 85.8% chr14 - 47712914 47713075 162 browser details YourSeq 110 1303 1479 3000 81.1% chr13 + 57121249 57121408 160 browser details YourSeq 108 1325 1479 3000 82.9% chr9 + 94642623 94642764 142 browser details YourSeq 104 1300 1472 3000 86.6% chr14 - 56089056 56089226 171 browser details YourSeq 103 1334 1465 3000 88.1% chr11 + 121093516 121093645 130 browser details YourSeq 103 1341 1487 3000 82.2% chr11 + 8820872 8821012 141 browser details YourSeq 100 1323 1465 3000 82.7% chr11 - 83338300 83338429 130 browser details YourSeq 100 1323 1469 3000 83.1% chr11 + 97425330 97425468 139 browser details YourSeq 99 1345 1474 3000 89.7% chr13 - 98997767 99071364 73598 browser details YourSeq 99 1303 1465 3000 78.6% chr10 + 8920094 8920229 136 browser details YourSeq 99 1321 1479 3000 84.2% chr1 + 128059044 128059191 148 browser details YourSeq 98 1324 1465 3000 83.5% chr11 - 85651308 85651435 128 browser details YourSeq 95 1331 1468 3000 87.8% chr11 - 90673250 90673386 137 browser details YourSeq 95 1336 1465 3000 84.0% chr11 - 20345224 20345349 126 browser details YourSeq 94 1342 1474 3000 82.9% chr6 - 87472569 87472697 129 browser details YourSeq 94 1297 1452 3000 79.4% chr11 - 96754288 96754419 132 browser details YourSeq 93 1324 1465 3000 82.8% chr16 - 10253194 10253322 129

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cutc cutC copper transporter [ Mus musculus (house mouse) ] Gene ID: 66388, updated on 12-Aug-2019

Gene summary

Official Symbol Cutc provided by MGI Official Full Name cutC copper transporter provided by MGI Primary source MGI:MGI:1913638 See related Ensembl:ENSMUSG00000025193 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 CGI-32; AI326282; 2310039I18Rik Expression Ubiquitous expression in testis adult (RPKM 6.5), bladder adult (RPKM 4.1) and 28 other tissues See more Orthologs human all

Genomic context

Location: 19; 19 C3 See Cutc in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (43753023..43768638)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (43827513..43843128)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 4 transcripts

Gene: Cutc ENSMUSG00000025193

Description cutC copper transporter [Source:MGI Symbol;Acc:MGI:1913638] Gene Synonyms 2310039I18Rik, CGI-32 Location Chromosome 19: 43,752,996-43,768,638 forward strand. GRCm38:CM001012.2 About this gene This gene has 4 transcripts (splice variants), 192 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cutc-201 ENSMUST00000026199.13 1298 262aa ENSMUSP00000026199.7 Protein coding CCDS29837 F8WHX2 TSL:1 GENCODE basic

Cutc-202 ENSMUST00000112047.9 1233 272aa ENSMUSP00000107678.3 Protein coding CCDS50443 Q9D8X1 TSL:1 GENCODE basic APPRIS P1

Cutc-204 ENSMUST00000153295.1 788 254aa ENSMUSP00000118906.1 Protein coding - D3YY50 CDS 3' incomplete TSL:3

Cutc-203 ENSMUST00000123564.1 685 No protein - Retained intron - - TSL:3

35.64 kb Forward strand 43.75Mb 43.76Mb 43.77Mb Genes Cutc-201 >protein coding (Comprehensive set...

Cutc-202 >protein coding

Cutc-204 >protein coding

Cutc-203 >retained intron

Contigs < AC141888.4 Genes < Cox15-201protein coding (Comprehensive set...

Regulatory Build

43.75Mb 43.76Mb 43.77Mb Reverse strand 35.64 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000112047

15.54 kb Forward strand

Cutc-202 >protein coding

ENSMUSP00000107... Superfamily Copper homeostasis (CutC) domain superfamily Pfam Copper homeostasis CutC domain PANTHER Copper homeostasis protein CutC HAMAP Copper homeostasis protein CutC Gene3D Copper homeostasis (CutC) domain superfamily

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 272

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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