https://www.alphaknockout.com

Mouse Ccnc Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Ccnc gene (NCBI Reference Sequence: NM_001290422 ; Ensembl: ENSMUSG00000028252 ) is located on Mouse chromosome 4. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000065928). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ccnc gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-369K6 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: Homozygous null mice die prenatally and exhibit growth retardation and placental defects.

Exon 2 starts from about 3.9% of the coding region. The knockout of Exon 2~4 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 2481 bp, and the size of intron 4 for 3'-loxP site insertion: 2858 bp. The size of effective cKO region: ~2662 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 4 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ccnc 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(9162bp) | A(28.76% 2635) | C(19.05% 1745) | T(30.89% 2830) | G(21.31% 1952)

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% chr4 + 21727148 21730147 3000 browser details YourSeq 52 1319 1430 3000 93.4% chr5 - 60300538 60300651 114 browser details YourSeq 50 1359 1438 3000 79.4% chr13 - 61534540 61534612 73 browser details YourSeq 50 1359 1438 3000 77.7% chr12 + 82015625 82015697 73 browser details YourSeq 45 1147 1438 3000 64.8% chr1 + 54954216 54954353 138 browser details YourSeq 44 1383 1438 3000 87.3% chr18 + 33555934 33555984 51 browser details YourSeq 44 2869 2969 3000 95.9% chr12 + 51613587 51614129 543 browser details YourSeq 43 1359 1438 3000 81.3% chrX + 63211625 63211697 73 browser details YourSeq 38 1388 1438 3000 83.4% chr4 + 126389493 126389538 46 browser details YourSeq 38 2907 2999 3000 75.9% chr18 + 46338315 46338405 91 browser details YourSeq 37 1395 1438 3000 93.1% chr11 - 12839044 12839088 45 browser details YourSeq 37 1348 1400 3000 86.3% chr1 + 26447345 26447407 63 browser details YourSeq 35 1380 1438 3000 94.9% chrX + 60170238 60170817 580 browser details YourSeq 35 1835 1878 3000 90.0% chr1 + 7888781 7888823 43 browser details YourSeq 34 1380 1427 3000 94.8% chr5 - 124409151 124409538 388 browser details YourSeq 33 1388 1438 3000 78.4% chr19 - 36922524 36922567 44 browser details YourSeq 32 1388 1427 3000 82.4% chr5 + 140694515 140694549 35 browser details YourSeq 31 2907 2964 3000 87.9% chrX + 152397776 152397831 56 browser details YourSeq 29 1359 1401 3000 87.1% chr11 - 96236544 96236584 41 browser details YourSeq 29 1359 1401 3000 83.8% chr10 + 52144970 52145012 43

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% chr4 + 21732810 21735809 3000 browser details YourSeq 87 662 834 3000 80.5% chr10 + 103595519 103595685 167 browser details YourSeq 84 1575 1859 3000 92.0% chr1 + 36058383 36058775 393 browser details YourSeq 81 667 808 3000 78.7% chr12 + 31383906 31384038 133 browser details YourSeq 80 680 808 3000 80.4% chr10 + 70768712 70768833 122 browser details YourSeq 75 685 825 3000 84.0% chr1 + 120751367 120751508 142 browser details YourSeq 72 668 822 3000 82.4% chr15 - 79415555 79415696 142 browser details YourSeq 71 700 834 3000 85.9% chr7 - 144431776 144431922 147 browser details YourSeq 71 669 836 3000 80.0% chr11 + 86393056 86393193 138 browser details YourSeq 67 330 443 3000 81.9% chr5 - 33225534 33225621 88 browser details YourSeq 67 672 808 3000 80.3% chr5 - 30983704 30983833 130 browser details YourSeq 66 167 392 3000 92.5% chrX + 74216579 74216805 227 browser details YourSeq 66 644 762 3000 84.4% chr12 + 30757071 30757625 555 browser details YourSeq 65 667 807 3000 84.0% chr10 - 69333013 69333142 130 browser details YourSeq 64 700 829 3000 75.3% chr9 + 100974665 100974777 113 browser details YourSeq 63 176 393 3000 88.9% chr1 - 11207956 11208177 222 browser details YourSeq 63 669 770 3000 79.3% chr1 + 84570440 84570532 93 browser details YourSeq 62 680 773 3000 79.1% chr1 - 73753982 73754068 87 browser details YourSeq 61 672 808 3000 82.8% chr10 - 59440769 59440902 134 browser details YourSeq 61 342 478 3000 78.9% chr4 + 99927319 99927423 105

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

Page 4 of 8 https://www.alphaknockout.com

Gene and protein information: Ccnc cyclin C [ Mus musculus (house mouse) ] Gene ID: 51813, updated on 14-Aug-2019

Gene summary

Official Symbol Ccnc provided by MGI Official Full Name cyclin C provided by MGI Primary source MGI:MGI:1858199 See related Ensembl:ENSMUSG00000028252 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 CG1C; AI451004; AU020987 Expression Ubiquitous expression in CNS E18 (RPKM 5.3), CNS E14 (RPKM 4.7) and 28 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 A3 See Ccnc in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (21727701..21759922)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (21654850..21677693)

Chromosome 4 - NC_000070.6

Page 5 of 8 https://www.alphaknockout.com

Transcript information: This gene has 8 transcripts

Gene: Ccnc ENSMUSG00000028252

Description cyclin C [Source:MGI Symbol;Acc:MGI:1858199] Location Chromosome 4: 21,727,701-21,759,922 forward strand. GRCm38:CM000997.2 About this gene This gene has 8 transcripts (splice variants), 206 orthologues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ccnc-201 ENSMUST00000065928.10 3484 282aa ENSMUSP00000069076.4 Protein coding CCDS51127 Q8CAS3 TSL:1 GENCODE basic APPRIS ALT1

Ccnc-202 ENSMUST00000102997.7 3365 283aa ENSMUSP00000100062.1 Protein coding CCDS17998 Q62447 TSL:1 GENCODE basic APPRIS P3

Ccnc-204 ENSMUST00000120679.7 1453 266aa ENSMUSP00000113682.1 Protein coding CCDS71350 Q3UXL9 TSL:1 GENCODE basic

Ccnc-203 ENSMUST00000108240.2 1406 282aa ENSMUSP00000103875.2 Protein coding CCDS51127 Q8CAS3 TSL:1 GENCODE basic APPRIS ALT1

Ccnc-205 ENSMUST00000123294.7 1404 No protein - lncRNA - - TSL:1

Ccnc-207 ENSMUST00000133712.1 1247 No protein - lncRNA - - TSL:1

Ccnc-206 ENSMUST00000133597.7 1211 No protein - lncRNA - - TSL:1

Ccnc-208 ENSMUST00000145288.7 445 No protein - lncRNA - - TSL:1

Page 6 of 8 https://www.alphaknockout.com

52.22 kb Forward strand 21.72Mb 21.73Mb 21.74Mb 21.75Mb 21.76Mb Genes (Comprehensive set... Ccnc-201 >protein coding Usp45-208 >lncRNA

Ccnc-204 >protein coding

Ccnc-202 >protein coding

Ccnc-206 >lncRNA

Ccnc-205 >lncRNA

Ccnc-203 >protein coding

Ccnc-208 >lncRNA

Ccnc-207 >lncRNA

Contigs AL772187.6 > Genes < Tstd3-201protein coding (Comprehensive set...

Regulatory Build

21.72Mb 21.73Mb 21.74Mb 21.75Mb 21.76Mb Reverse strand 52.22 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000065928

22.85 kb Forward strand

Ccnc-201 >protein coding

ENSMUSP00000069... MobiDB lite Superfamily Cyclin-like superfamily SMART Cyclin-like Pfam Cyclin, N-terminal Cyclin, C-terminal domain 2

PIRSF PIRSF028758 PANTHER PTHR10026

Cyclin C/cyclin-like subunit Ssn8 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 80 120 160 200 240 282

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

Page 8 of 8