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Mouse Ccdc183 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ccdc183 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ccdc183 gene (NCBI Reference Sequence: NM_029859 ; Ensembl: ENSMUSG00000026940 ) is located on Mouse chromosome 2. 14 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 14 (Transcript: ENSMUST00000028309). Exon 7~10 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ccdc183 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-158L20 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 7 starts from about 41.64% of the coding region. The knockout of Exon 7~10 will result in frameshift of the gene. The size of intron 6 for 5'-loxP site insertion: 1363 bp, and the size of intron 10 for 3'-loxP site insertion: 676 bp. The size of effective cKO region: ~2225 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 6 7 8 9 10 11 12 13 14 1 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ccdc183 Homology arm cKO region Exon of mouse Rabl6 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(8725bp) | A(25.23% 2201) | C(27.0% 2356) | T(22.76% 1986) | G(25.01% 2182)

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.

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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% chr2 - 25612445 25615444 3000 browser details YourSeq 176 1258 2590 3000 93.7% chr11 - 88888133 89351896 463764 browser details YourSeq 154 1286 2592 3000 90.3% chr10 - 60897073 61284799 387727 browser details YourSeq 118 1263 1395 3000 97.6% chr19 - 42286449 42286629 181 browser details YourSeq 114 1263 1397 3000 95.3% chr12 + 116482396 116482574 179 browser details YourSeq 112 1265 1397 3000 95.2% chr16 - 55848237 55848697 461 browser details YourSeq 111 1263 1397 3000 97.5% chr2 - 155924981 155925350 370 browser details YourSeq 110 1303 2569 3000 92.4% chr11 + 97586504 97882416 295913 browser details YourSeq 104 2505 2640 3000 91.3% chr17 - 78549207 78549354 148 browser details YourSeq 103 2503 2639 3000 88.8% chr5 - 74090445 74090602 158 browser details YourSeq 103 1284 1400 3000 94.1% chr12 + 76208520 76208636 117 browser details YourSeq 102 1286 1397 3000 96.5% chr11 + 23112980 23113292 313 browser details YourSeq 101 1284 1398 3000 94.0% chr5 - 88666257 88666371 115 browser details YourSeq 101 1286 1396 3000 95.5% chr11 - 87199402 87199512 111 browser details YourSeq 101 1265 1399 3000 94.7% chr11 - 57339862 57340036 175 browser details YourSeq 101 1285 1397 3000 92.6% chr10 - 114596758 114596866 109 browser details YourSeq 101 1284 1394 3000 95.5% chr2 + 20864077 20864187 111 browser details YourSeq 101 1284 1400 3000 92.6% chr15 + 75958313 75958425 113 browser details YourSeq 100 1285 1397 3000 93.3% chr4 - 71031374 71031482 109 browser details YourSeq 100 1281 1397 3000 90.5% chr3 - 153613005 153613119 115

Note: The 3000 bp section upstream of Exon 7 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% chr2 - 25607220 25610219 3000 browser details YourSeq 175 31 404 3000 82.5% chr10 - 93051804 93052054 251 browser details YourSeq 152 117 408 3000 89.3% chr2 + 171505688 171506262 575 browser details YourSeq 152 48 405 3000 93.8% chr1 + 33600192 33601782 1591 browser details YourSeq 140 91 405 3000 92.7% chr1 + 33600102 33600621 520 browser details YourSeq 129 91 404 3000 93.9% chr1 + 33600192 33600884 693 browser details YourSeq 123 56 412 3000 94.5% chr13 + 79686008 79686452 445 browser details YourSeq 122 91 405 3000 93.6% chr1 + 33600543 33601838 1296 browser details YourSeq 108 121 337 3000 91.2% chr1 - 58654537 58654894 358 browser details YourSeq 105 245 422 3000 97.3% chr2 - 25610075 25610303 229 browser details YourSeq 102 91 356 3000 94.8% chr1 + 33600577 33601863 1287 browser details YourSeq 100 111 404 3000 84.3% chr1 - 33617366 33617614 249 browser details YourSeq 95 91 401 3000 84.3% chr1 - 33617282 33617555 274 browser details YourSeq 94 91 405 3000 94.4% chr1 + 33600481 33601863 1383 browser details YourSeq 94 114 405 3000 95.3% chr1 + 33600102 33600773 672 browser details YourSeq 86 166 410 3000 93.0% chr13 - 57658644 57658961 318 browser details YourSeq 86 68 407 3000 94.2% chr13 + 42084766 42085261 496 browser details YourSeq 86 91 330 3000 94.8% chr1 + 33601434 33601863 430 browser details YourSeq 80 33 307 3000 93.7% chr13 - 77920543 77920878 336 browser details YourSeq 79 206 385 3000 78.0% chr1 + 66528171 66528261 91

Note: The 3000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ccdc183 coiled-coil domain containing 183 [ Mus musculus (house mouse) ] Gene ID: 77058, updated on 12-Aug-2019

Gene summary

Official Symbol Ccdc183 provided by MGI Official Full Name coiled-coil domain containing 183 provided by MGI Primary source MGI:MGI:1924308 See related Ensembl:ENSMUSG00000026940 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 Cccd183; Kiaa1984; 4921530D09Rik Expression Restricted expression toward testis adult (RPKM 44.3) See more Orthologs human all

Genomic context

Location: 2; 2 A3 See Ccdc183 in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (25608629..25617678, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (25464149..25473198, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 2 transcripts

Gene: Ccdc183 ENSMUSG00000026940

Description coiled-coil domain containing 183 [Source:MGI Symbol;Acc:MGI:1924308] Gene Synonyms 4921530D09Rik, Cccd183 Location Chromosome 2: 25,608,635-25,617,678 reverse strand. GRCm38:CM000995.2 About this gene This gene has 2 transcripts (splice variants), 43 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ccdc183-201 ENSMUST00000028309.3 1719 534aa ENSMUSP00000028309.3 Protein coding CCDS50533 A2AJB1 TSL:5 GENCODE basic APPRIS P1

Ccdc183-202 ENSMUST00000129520.1 2958 No protein - lncRNA - - TSL:2

29.04 kb Forward strand 25.60Mb 25.61Mb 25.62Mb Genes Fcnaos-203 >lncRNA (Comprehensive set...

Fcnaos-201 >lncRNA

Fcnaos-204 >lncRNA

Contigs AL732590.7 > Genes (Comprehensive set... < Rabl6-201protein coding < Ccdc183-201protein coding < Tmem141-205protein codin

< Rabl6-208lncRNA < Ccdc183-202lncRNA < Tmem141-201protein coding< Fcna-202lncRNA

< Rabl6-204lncRNA < Tmem141-206lncRNA

< Rabl6-206lncRNA < Tmem141-202lncRNA

< Tmem141-204lncRNA

< Tmem141-203lncRNA

< Tmem141-207lncRNA

Regulatory Build

25.60Mb 25.61Mb 25.62Mb Reverse strand 29.04 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000028309

< Ccdc183-201protein coding

Reverse strand 9.04 kb

ENSMUSP00000028... Low complexity (Seg) Coiled-coils (Ncoils) PANTHER PTHR47115

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

Variant Legend

missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 534

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

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