https://www.alphaknockout.com Mouse Ccni Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ccni conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ccni gene (NCBI Reference Sequence: NM_017367 ; Ensembl: ENSMUSG00000063015 ) is located on Mouse chromosome 5. 7 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 7 (Transcript: ENSMUST00000058550). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ccni gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-141M16 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 targeted null mutation are viable and fertile and do not display any gross physical or behavioral abnormalities. Exon 2 starts from about 100% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 3451 bp, and the size of intron 2 for 3'-loxP site insertion: 12612 bp. The size of effective cKO region: ~614 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 gRNA region 5' gRNA region 3' 1 2 7 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ccni 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. 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(7114bp) | A(29.81% 2121) | C(18.3% 1302) | T(31.75% 2259) | G(20.13% 1432) 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% chr5 - 93202669 93205668 3000 browser details YourSeq 33 2344 2378 3000 97.2% chr4 + 107982277 107982311 35 browser details YourSeq 32 2345 2388 3000 86.4% chr13 + 40170284 40170327 44 browser details YourSeq 31 2321 2371 3000 80.4% chr17 - 29141306 29141356 51 browser details YourSeq 31 2321 2379 3000 83.0% chr4 + 58135856 58135913 58 browser details YourSeq 29 2321 2387 3000 71.7% chr13 - 47891170 47891236 67 browser details YourSeq 29 2359 2391 3000 94.0% chr8 + 95949049 95949081 33 browser details YourSeq 28 132 167 3000 93.8% chr14 + 116057776 116057812 37 browser details YourSeq 27 139 167 3000 89.3% chr15 - 20079621 20079648 28 browser details YourSeq 27 2357 2387 3000 93.6% chr13 - 63932151 63932181 31 browser details YourSeq 24 2649 2680 3000 87.5% chr15 + 24192766 24192797 32 browser details YourSeq 23 139 163 3000 87.5% chr11 - 61487289 61487312 24 browser details YourSeq 22 2368 2391 3000 95.9% chr5 - 124581565 124581588 24 browser details YourSeq 22 2321 2370 3000 72.0% chr15 - 12102787 12102836 50 browser details YourSeq 22 2354 2377 3000 95.9% chr4 + 116131234 116131257 24 browser details YourSeq 22 2458 2479 3000 100.0% chr3 + 138739953 138739974 22 browser details YourSeq 20 145 164 3000 100.0% chr14 - 56397670 56397689 20 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% chr5 - 93199055 93202054 3000 browser details YourSeq 166 1658 1858 3000 91.8% chr16 + 56084530 56084728 199 browser details YourSeq 165 1672 1857 3000 95.1% chr1 + 45798692 45798887 196 browser details YourSeq 164 1671 1859 3000 95.2% chr9 - 7649267 7649462 196 browser details YourSeq 164 1673 1856 3000 95.1% chr6 - 34341594 34341780 187 browser details YourSeq 164 1690 1943 3000 93.2% chr11 - 98013598 98014002 405 browser details YourSeq 163 1673 1858 3000 96.1% chr2 - 130834523 130834710 188 browser details YourSeq 163 1675 1860 3000 95.1% chr14 - 64662788 64662978 191 browser details YourSeq 163 1676 1859 3000 94.6% chr6 + 51264483 51264670 188 browser details YourSeq 163 1671 1859 3000 93.6% chr12 + 108299973 108300164 192 browser details YourSeq 162 1669 1858 3000 93.1% chr10 - 44326365 44326564 200 browser details YourSeq 162 1672 1859 3000 95.6% chr15 + 69045981 69046171 191 browser details YourSeq 161 1671 1858 3000 95.0% chr13 + 104263713 104263904 192 browser details YourSeq 160 1671 1858 3000 93.1% chr4 - 108323094 108323300 207 browser details YourSeq 160 1672 1858 3000 94.0% chr12 - 85014239 85014425 187 browser details YourSeq 160 1679 1858 3000 92.6% chr8 + 106311644 106311818 175 browser details YourSeq 160 1667 1858 3000 92.2% chr4 + 150611183 150611379 197 browser details YourSeq 160 1677 1858 3000 95.1% chr15 + 102354287 102354481 195 browser details YourSeq 159 1682 1859 3000 95.0% chrX - 52784348 53000621 216274 browser details YourSeq 159 1671 1856 3000 94.0% chr12 - 76834324 76834510 187 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Ccni cyclin I [ Mus musculus (house mouse) ] Gene ID: 12453, updated on 12-Aug-2019 Gene summary Official Symbol Ccni provided by MGI Official Full Name cyclin I provided by MGI Primary source MGI:MGI:1341077 See related Ensembl:ENSMUSG00000063015 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 Expression Ubiquitous expression in testis adult (RPKM 56.6), colon adult (RPKM 53.2) and 28 other tissues See more Orthologs human all Genomic context Location: 5; 5 E2 See Ccni in Genome Data Viewer Exon count: 8 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (93181933..93206495, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (93610959..93635521, complement) Chromosome 5 - NC_000071.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Ccni ENSMUSG00000063015 Description cyclin I [Source:MGI Symbol;Acc:MGI:1341077] Location Chromosome 5: 93,181,933-93,206,495 reverse strand. GRCm38:CM000998.2 About this gene This gene has 5 transcripts (splice variants), 236 orthologues, 16 paralogues, 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 Ccni- ENSMUST00000058550.14 2804 377aa ENSMUSP00000050189.8 Protein coding CCDS19436 Q9Z2V9 TSL:1 201 GENCODE basic APPRIS P1 Ccni- ENSMUST00000144514.2 1068 81aa ENSMUSP00000122434.1 Protein coding - D3Z602 CDS 3' incomplete 203 TSL:5 Ccni- ENSMUST00000151568.7 658 172aa ENSMUSP00000116224.1 Protein coding - D3Z680 CDS 3' incomplete 204 TSL:5 Ccni- ENSMUST00000201823.3 446 148aa ENSMUSP00000143972.1 Protein coding - A0A0J9YU25 CDS 5' and 3' 205 incomplete TSL:3 Ccni- ENSMUST00000123033.1 507 No - Retained - - TSL:2 202 protein intron Page 6 of 8 https://www.alphaknockout.com 44.56 kb Forward strand 93.18Mb 93.19Mb 93.20Mb 93.21Mb Genes Sept11-201 >protein coding Gm2986-201 >processed pseudogene 2010109A12Rik-201 >protein coding (Comprehensive set... Sept11-208 >protein coding 2010109A12Rik-202 >retained intron Sept11-203 >protein coding Sept11-207 >protein coding Gm43682-201 >TEC Contigs < AC134827.4 Genes < Ccni-201protein coding (Comprehensive set... < Ccni-205protein coding < Gm43681-201TEC < Ccni-203protein coding < Ccni-202retained intron < Ccni-204protein coding Regulatory Build 93.18Mb 93.19Mb 93.20Mb 93.21Mb Reverse strand 44.56 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000058550 < Ccni-201protein coding Reverse strand 24.56 kb ENSMUSP00000050... Superfamily Cyclin-like superfamily SMART Cyclin-like Pfam Cyclin, N-terminal PIRSF Cyclin PANTHER Cyclin I Cyclin Gene3D 1.10.472.10 CDD Cyclin-like All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 377 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.