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Mouse Cfap206 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cfap206 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cfap206 gene (NCBI Reference Sequence: NM_027041 ; Ensembl: ENSMUSG00000028294 ) is located on Mouse chromosome 4. 11 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000108136). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cfap206 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-98N9 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 4 starts from about 12.76% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2999 bp, and the size of intron 4 for 3'-loxP site insertion: 2018 bp. The size of effective cKO region: ~591 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 4 5 11 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cfap206 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. 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(7091bp) | A(27.19% 1928) | C(21.14% 1499) | T(30.25% 2145) | G(21.42% 1519)

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 - 34725156 34728155 3000 browser details YourSeq 884 665 2111 3000 89.5% chr4 - 95172758 95174023 1266 browser details YourSeq 873 542 1642 3000 91.4% chr18 - 32468944 32470072 1129 browser details YourSeq 865 564 1675 3000 90.6% chrX + 153332791 153334061 1271 browser details YourSeq 862 563 1682 3000 91.4% chr19 - 29766344 29767465 1122 browser details YourSeq 860 565 1682 3000 90.4% chrX + 14425659 14426773 1115 browser details YourSeq 858 560 1646 3000 90.9% chr14 + 17854965 17856095 1131 browser details YourSeq 851 558 1659 3000 91.2% chr2 + 81061086 81062233 1148 browser details YourSeq 849 561 1678 3000 91.3% chrX + 85405444 85406570 1127 browser details YourSeq 846 562 1682 3000 90.9% chr6 - 23698902 23700102 1201 browser details YourSeq 838 553 1682 3000 91.2% chr6 + 48879182 48880531 1350 browser details YourSeq 834 564 1682 3000 89.8% chr7 + 23344930 23346080 1151 browser details YourSeq 834 560 1682 3000 91.0% chr2 + 50258610 50259742 1133 browser details YourSeq 833 575 1891 3000 90.6% chr17 + 53141746 53143106 1361 browser details YourSeq 828 563 1682 3000 91.3% chr14 + 12031086 12032234 1149 browser details YourSeq 817 587 1682 3000 89.8% chrX + 60011703 60012796 1094 browser details YourSeq 816 1684 2697 3000 91.0% chr12 + 79415075 79416093 1019 browser details YourSeq 814 579 1682 3000 89.8% chr12 - 94241414 94242559 1146 browser details YourSeq 814 577 1664 3000 89.9% chr7 + 111815896 111817014 1119 browser details YourSeq 811 563 1682 3000 89.5% chr7 + 50835622 50836757 1136

Note: The 3000 bp section upstream of Exon 4 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 - 34721565 34724564 3000 browser details YourSeq 97 1056 1362 3000 90.0% chr1 - 92381870 92531005 149136 browser details YourSeq 96 999 1346 3000 91.3% chr10 - 79662324 79662835 512 browser details YourSeq 95 809 1375 3000 80.6% chr12 - 87067208 87067652 445 browser details YourSeq 95 1010 1285 3000 88.0% chr10 + 128419955 128420425 471 browser details YourSeq 94 1116 1346 3000 89.9% chr6 + 31657704 31657987 284 browser details YourSeq 92 1101 1335 3000 91.2% chr11 + 65567056 65567311 256 browser details YourSeq 90 1124 1373 3000 80.5% chr16 - 33408466 33408709 244 browser details YourSeq 90 805 1361 3000 77.0% chr15 - 58431868 58432266 399 browser details YourSeq 89 1114 1376 3000 78.8% chr3 - 36837027 36837287 261 browser details YourSeq 88 1101 1375 3000 88.0% chr13 + 21393484 21477348 83865 browser details YourSeq 86 1134 1371 3000 89.0% chr12 - 86457398 86457658 261 browser details YourSeq 80 1104 1335 3000 88.5% chr3 - 135396121 135396371 251 browser details YourSeq 79 810 1372 3000 78.5% chr2 - 92137811 92138304 494 browser details YourSeq 79 1120 1335 3000 78.2% chr16 - 22902009 22902253 245 browser details YourSeq 76 1041 1165 3000 81.6% chr2 - 155106505 155106631 127 browser details YourSeq 76 1121 1284 3000 86.6% chr11 + 76441227 76441417 191 browser details YourSeq 75 1132 1335 3000 86.6% chr10 - 63131549 63131768 220 browser details YourSeq 75 1137 1347 3000 77.1% chr14 + 16564092 16564286 195 browser details YourSeq 73 1258 1685 3000 69.8% chr6 + 86024854 86025007 154

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cfap206 cilia and flagella associated protein 206 [ Mus musculus (house mouse) ] Gene ID: 69329, updated on 12-Aug-2019

Gene summary

Official Symbol Cfap206 provided by MGI Official Full Name cilia and flagella associated protein 206 provided by MGI Primary source MGI:MGI:1916579 See related Ensembl:ENSMUSG00000028294 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 AU024269; 1700003M02Rik Expression Restricted expression toward testis adult (RPKM 129.5) See more Orthologs human all

Genomic context

Location: 4; 4 A5 See Cfap206 in Genome Data Viewer

Exon count: 13

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (34688574..34730263, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (34658581..34677455, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 6 transcripts

Gene: Cfap206 ENSMUSG00000028294

Description cilia and flagella associated protein 206 [Source:MGI Symbol;Acc:MGI:1916579] Gene Synonyms 1700003M02Rik Location Chromosome 4: 34,688,559-34,730,206 reverse strand. GRCm38:CM000997.2 About this gene This gene has 6 transcripts (splice variants), 223 orthologues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cfap206- ENSMUST00000029971.11 2246 622aa ENSMUSP00000029971.5 Protein coding CCDS84713 Q6PE87 TSL:5 201 GENCODE basic APPRIS P1

Cfap206- ENSMUST00000108136.7 1688 504aa ENSMUSP00000103771.1 Protein coding CCDS18031 Q6PE87 TSL:1 202 GENCODE basic

Cfap206- ENSMUST00000137514.2 765 210aa ENSMUSP00000116947.1 Protein coding - Z4YM47 CDS 3' 204 incomplete TSL:5

Cfap206- ENSMUST00000162495.7 628 46aa ENSMUSP00000124760.1 Nonsense mediated - E0CXC1 TSL:3 206 decay

Cfap206- ENSMUST00000135563.1 690 No - lncRNA - - TSL:5 203 protein

Cfap206- ENSMUST00000160209.1 557 No - lncRNA - - TSL:3 205 protein

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61.65 kb Forward strand 34.68Mb 34.70Mb 34.72Mb 34.74Mb Genes Gm22849-201 >misc RNA (Comprehensive set...

Contigs AL928738.8 > Genes (Comprehensive set... < Slc35a1-201protein coding < Cfap206-201protein coding

< Slc35a1-204lncRNA < Cfap206-203lncRNA < Cfap206-206nonsense mediated decay

< Slc35a1-203protein coding < Cfap206-202protein coding

< Slc35a1-202lncRNA < Cfap206-204protein coding

< Slc35a1-205lncRNA < Cfap206-205lncRNA

Regulatory Build

34.68Mb 34.70Mb 34.72Mb 34.74Mb Reverse strand 61.65 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000108136

< Cfap206-202protein coding

Reverse strand 18.85 kb

ENSMUSP00000103... Coiled-coils (Ncoils) Pfam Cilia- and flagella-associated protein 206 PANTHER Cilia- and flagella-associated protein 206

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 504

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

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