https://www.alphaknockout.com Mouse Ccny Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ccny conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ccny gene (NCBI Reference Sequence: NM_026484.3 ; Ensembl: ENSMUSG00000024286 ) is located on Mouse chromosome 18. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000053917). Exon 6~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ccny gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-444I9 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 impaired adipogenesis and lipid production. Exon 6 starts from about 39.3% of the coding region. The knockout of Exon 6~7 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 3916 bp, and the size of intron 7 for 3'-loxP site insertion: 12243 bp. The size of effective cKO region: ~778 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 6 7 10 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ccny Homology arm cKO region loxP site Page 2 of 7 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. 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(7278bp) | A(27.64% 2012) | C(19.95% 1452) | T(31.92% 2323) | G(20.49% 1491) 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. Page 3 of 7 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% chr18 - 9345722 9348721 3000 browser details YourSeq 88 2033 2224 3000 88.0% chr9 + 61188981 61189187 207 browser details YourSeq 73 2074 2227 3000 93.0% chrX + 13753024 13753192 169 browser details YourSeq 72 2055 2224 3000 89.1% chr9 - 49561758 49561948 191 browser details YourSeq 72 2060 2207 3000 92.0% chr19 - 26360921 26361081 161 browser details YourSeq 66 2049 2202 3000 91.3% chr13 + 73045178 73045343 166 browser details YourSeq 64 2049 2224 3000 82.0% chr10 + 60627887 60628081 195 browser details YourSeq 59 2043 2224 3000 94.1% chrX - 161953890 161954080 191 browser details YourSeq 58 2052 2379 3000 91.5% chr14 + 9241590 9242899 1310 browser details YourSeq 49 2170 2270 3000 94.7% chr11 + 19635967 19636070 104 browser details YourSeq 47 2169 2225 3000 87.5% chr15 - 58812801 58812856 56 browser details YourSeq 45 2190 2271 3000 94.2% chr4 - 125935622 125935705 84 browser details YourSeq 45 2033 2104 3000 84.3% chr15 - 86505876 86505945 70 browser details YourSeq 44 2075 2194 3000 94.0% chr10 + 90538959 90539096 138 browser details YourSeq 43 2173 2227 3000 86.8% chr4 - 65199289 65199342 54 browser details YourSeq 41 2045 2093 3000 91.9% chr4 + 82840085 82840133 49 browser details YourSeq 40 2049 2103 3000 91.7% chr11 - 48523005 48523064 60 browser details YourSeq 40 21 144 3000 80.9% chr11 + 54976305 54976423 119 browser details YourSeq 40 2816 2995 3000 63.3% chr1 + 87987601 87987691 91 browser details YourSeq 39 2173 2227 3000 83.1% chr1 + 121148320 121148373 54 Note: The 3000 bp section upstream of Exon 6 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% chr18 - 9341944 9344943 3000 browser details YourSeq 1573 919 2962 3000 90.8% chr1 - 189467192 190023777 556586 browser details YourSeq 1475 962 2962 3000 89.2% chr15 - 36358270 36360324 2055 browser details YourSeq 1473 924 2962 3000 88.6% chr15 + 34626648 35249920 623273 browser details YourSeq 1455 927 2962 3000 88.9% chr10 - 40014410 40016434 2025 browser details YourSeq 1454 944 2962 3000 89.7% chr17 - 85423800 85425827 2028 browser details YourSeq 1444 908 2962 3000 88.7% chr17 - 94158862 94160906 2045 browser details YourSeq 1441 929 2962 3000 88.2% chr3 + 115056732 115058750 2019 browser details YourSeq 1438 913 2951 3000 89.2% chr1 + 161512696 161514735 2040 browser details YourSeq 1430 928 2962 3000 87.9% chr18 - 52704268 52706283 2016 browser details YourSeq 1398 922 2962 3000 87.1% chr9 - 34320183 34322173 1991 browser details YourSeq 1388 922 2962 3000 88.5% chr2 + 13618677 13620705 2029 browser details YourSeq 1381 978 2962 3000 87.7% chrX - 16865966 16867972 2007 browser details YourSeq 1378 922 2962 3000 87.7% chr1 - 92580071 92582039 1969 browser details YourSeq 1359 978 2963 3000 87.8% chrX - 155531838 155533843 2006 browser details YourSeq 1358 905 2917 3000 88.0% chr1 + 4171430 4173446 2017 browser details YourSeq 1357 1127 2962 3000 89.5% chr17 - 89193632 89195461 1830 browser details YourSeq 1349 923 2962 3000 88.9% chr17 + 56526839 56528967 2129 browser details YourSeq 1348 919 2944 3000 87.8% chr14 - 36382380 36384401 2022 browser details YourSeq 1338 922 2962 3000 87.9% chr8 + 105029576 105031540 1965 Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ccny cyclin Y [ Mus musculus (house mouse) ] Gene ID: 67974, updated on 26-Jun-2020 Gene summary Official Symbol Ccny provided by MGI Official Full Name cyclin Y provided by MGI Primary source MGI:MGI:1915224 See related Ensembl:ENSMUSG00000024286 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 1700025H17Rik; 3110050L10Rik; 4631402G10Rik; 5730405I09Rik Expression Ubiquitous expression in adrenal adult (RPKM 25.9), ovary adult (RPKM 20.4) and 28 other tissues See more Orthologs human all Genomic context Location: 18; 18 A1 See Ccny in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (9312304..9450154, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (9314042..9450148, complement) Chromosome 18 - NC_000084.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Ccny ENSMUSG00000024286 Description cyclin Y [Source:MGI Symbol;Acc:MGI:1915224] Gene Synonyms 1700025H17Rik, 3110050L10Rik, 4631402G10Rik, 5730405I09Rik Location Chromosome 18: 9,312,304-9,450,154 reverse strand. GRCm38:CM001011.2 About this gene This gene has 5 transcripts (splice variants), 114 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 19 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ccny- ENSMUST00000053917.5 5757 341aa ENSMUSP00000050001.4 Protein coding CCDS29050 Q8BGU5 TSL:1 201 GENCODE basic APPRIS P1 Ccny- ENSMUST00000234102.1 2505 316aa ENSMUSP00000157340.1 Protein coding - Q8BGU5 GENCODE 202 basic Ccny- ENSMUST00000234844.1 1354 99aa ENSMUSP00000157086.1 Nonsense mediated - A0A3Q4EBW6 - 205 decay Ccny- ENSMUST00000234779.1 1341 26aa ENSMUSP00000157353.1 Nonsense mediated - A0A3Q4EIH7 CDS 5' 204 decay incomplete Ccny- ENSMUST00000234382.1 1046 No - Retained intron - - - 203 protein 157.85 kb Forward strand 9.32Mb 9.34Mb 9.36Mb 9.38Mb 9.40Mb 9.42Mb 9.44Mb 9.46Mb Genes Gm50053-201 >processed pseudogene (Comprehensive set... Contigs < AC161530.4 Genes (Comprehensive set... < Ccny-201protein coding < Ccny-202protein coding < Ccny-204nonsense mediated decay < Gm50055-201TEC < Ccny-205nonsense mediated decay < Ccny-203retained intron Regulatory Build 9.32Mb 9.34Mb 9.36Mb 9.38Mb 9.40Mb 9.42Mb 9.44Mb 9.46Mb Reverse strand 157.85 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000053917 < Ccny-201protein coding Reverse strand 137.85 kb ENSMUSP00000050... Low complexity (Seg) Superfamily Cyclin-like superfamily SMART Cyclin-like Pfam Cyclin, N-terminal PIRSF Cyclin Y PANTHER PTHR14248:SF33 PTHR14248 Gene3D 1.10.472.10 CDD Cyclin-like All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 341 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.