https://www.alphaknockout.com Mouse Cfap298 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Cfap298 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cfap298 gene (NCBI Reference Sequence: NM_026502.2 ; Ensembl: ENSMUSG00000022972 ) is located on Mouse chromosome 16. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000023694). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cfap298 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-100B13 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 2 starts from about 16.09% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 3420 bp, and the size of intron 3 for 3'-loxP site insertion: 2407 bp. The size of effective cKO region: ~1746 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 7 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Cfap298 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com 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(8246bp) | A(23.7% 1954) | C(26.18% 2159) | T(24.53% 2023) | G(25.59% 2110) 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. Page 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr16 - 90931412 90934411 3000 browser details YourSeq 107 477 1040 3000 77.9% chr7 - 110291579 110291828 250 browser details YourSeq 100 469 608 3000 86.0% chr14 + 76370369 76370506 138 browser details YourSeq 89 1318 1753 3000 89.4% chr15 + 16463059 16463572 514 browser details YourSeq 88 718 1881 3000 94.0% chr18 - 38026820 38373404 346585 browser details YourSeq 84 468 584 3000 86.4% chr2 - 138964161 138964280 120 browser details YourSeq 84 476 584 3000 92.0% chr18 - 42458590 42458740 151 browser details YourSeq 83 521 609 3000 96.7% chr11 - 69725626 69725714 89 browser details YourSeq 80 830 1353 3000 90.0% chr17 + 15389102 15389685 584 browser details YourSeq 77 718 891 3000 88.2% chr9 - 94478028 94478202 175 browser details YourSeq 74 1138 1376 3000 90.3% chr10 + 76200830 76201103 274 browser details YourSeq 74 697 885 3000 90.2% chr1 + 125427229 125830354 403126 browser details YourSeq 71 718 843 3000 88.3% chr7 + 122899956 122900079 124 browser details YourSeq 71 718 891 3000 85.2% chr1 + 133938767 133938940 174 browser details YourSeq 70 718 892 3000 79.9% chr7 - 65247419 65247590 172 browser details YourSeq 68 697 880 3000 80.0% chrX - 11682428 11682610 183 browser details YourSeq 68 718 877 3000 88.9% chr18 - 14975098 14975256 159 browser details YourSeq 67 476 602 3000 86.1% chr15 - 101243278 101243403 126 browser details YourSeq 66 722 886 3000 86.6% chr14 - 62618820 62618983 164 browser details YourSeq 66 718 873 3000 86.6% chr7 + 117968819 117968972 154 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% chr16 - 90926666 90929665 3000 browser details YourSeq 73 285 472 3000 91.3% chr19 + 36070064 36070267 204 browser details YourSeq 67 257 462 3000 87.7% chr13 + 47457078 47457523 446 browser details YourSeq 63 1461 1602 3000 89.9% chr15 - 77426310 77426471 162 browser details YourSeq 49 428 479 3000 98.1% chr1 - 165787383 165787681 299 browser details YourSeq 48 420 471 3000 98.1% chr12 + 30735110 30735416 307 browser details YourSeq 47 1462 1552 3000 94.5% chr16 - 4575204 4575308 105 browser details YourSeq 45 359 439 3000 78.8% chr18 - 10259013 10259074 62 browser details YourSeq 43 304 388 3000 74.6% chr11 + 99987835 99987907 73 browser details YourSeq 42 1558 1615 3000 83.0% chr12 + 41064064 41064117 54 browser details YourSeq 39 728 775 3000 93.5% chr11 + 95408730 95408785 56 browser details YourSeq 38 723 774 3000 93.1% chr13 + 56704263 56704320 58 browser details YourSeq 38 1466 1633 3000 93.1% chr11 + 88836518 88836686 169 browser details YourSeq 37 725 778 3000 86.1% chr14 - 24729941 24729992 52 browser details YourSeq 37 1576 1631 3000 76.8% chr1 + 59189406 59189454 49 browser details YourSeq 36 1580 1627 3000 83.0% chr1 + 132393487 132393533 47 browser details YourSeq 35 725 774 3000 94.8% chr11 - 120839889 120839939 51 browser details YourSeq 35 1558 1602 3000 82.1% chr12 + 84324000 84324040 41 browser details YourSeq 33 311 388 3000 76.4% chr11 - 88273950 88274016 67 browser details YourSeq 33 362 431 3000 90.3% chr13 + 18231426 18231534 109 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Cfap298 cilia and flagella associate protien 298 [ Mus musculus (house mouse) ] Gene ID: 68001, updated on 26-Jun-2020 Gene summary Official Symbol Cfap298 provided by MGI Official Full Name cilia and flagella associate protien 298 provided by MGI Primary source MGI:MGI:1915251 See related Ensembl:ENSMUSG00000022972 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 1110004E09Rik Expression Ubiquitous expression in testis adult (RPKM 38.1), CNS E11.5 (RPKM 13.4) and 28 other tissues See more Genomic context Location: 16; 16 C3.3 See Cfap298 in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (90925811..90934849, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (90926056..90935094, complement) Chromosome 16 - NC_000082.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Cfap298 ENSMUSG00000022972 Description cilia and flagella associate protien 298 [Source:MGI Symbol;Acc:MGI:1915251] Gene Synonyms 1110004E09Rik Location Chromosome 16: 90,925,809-90,935,114 reverse strand. GRCm38:CM001009.2 About this gene This gene has 7 transcripts (splice variants), 268 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cfap298- ENSMUST00000023694.10 1430 290aa ENSMUSP00000023694.3 Protein coding CCDS28321 Q8BL95 TSL:1 201 GENCODE basic APPRIS P1 Cfap298- ENSMUST00000149833.7 597 198aa ENSMUSP00000123417.1 Protein coding - D3YZC4 CDS 3' 206 incomplete TSL:5 Cfap298- ENSMUST00000142340.1 560 150aa ENSMUSP00000120107.1 Protein coding - D3YUI6 CDS 3' 205 incomplete TSL:5 Cfap298- ENSMUST00000129345.7 489 156aa ENSMUSP00000122163.1 Protein coding - F7C0A6 CDS 5' 203 incomplete TSL:5 Cfap298- ENSMUST00000125519.7 1014 145aa ENSMUSP00000117691.1 Nonsense mediated - D6REH7 TSL:5 202 decay Cfap298- ENSMUST00000131583.1 374 No - Processed transcript - - TSL:3 204 protein Cfap298- ENSMUST00000231711.1 426 No - Retained intron - - - 207 protein Page 6 of 8 https://www.alphaknockout.com 29.31 kb Forward strand 90.92Mb 90.93Mb 90.94Mb Genes Gm49727-201 >TEC 4930534H18Rik-201 >bidirectional promoter lncRNA (Comprehensive set... Contigs < AC134560.5 Genes < Cfap298-201protein coding < Synj1-203protein coding (Comprehensive set... < Cfap298-203protein coding < Synj1-210protein coding < Cfap298-202nonsense mediated decay < Synj1-202protein coding < Cfap298-207retained intron < Synj1-208protein coding < Cfap298-206protein coding < Synj1-205processed transcript < Cfap298-205protein coding < Synj1-206protein coding < Cfap298-204processed transcript < Synj1-212protein coding < Synj1-204protein coding Regulatory Build 90.92Mb 90.93Mb 90.94Mb Reverse strand 29.31 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding Bidirectional promoter lncRNA processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000023694 < Cfap298-201protein coding Reverse strand 9.31 kb ENSMUSP00000023... Low complexity (Seg) Pfam Cilia- and flagella-associated protein 298 PANTHER PTHR13238:SF4 Cilia- and flagella-associated protein 298 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 290 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.