https://www.alphaknockout.com
Mouse Rcc2 Knockout Project (CRISPR/Cas9)
Objective: To create a Rcc2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Rcc2 gene (NCBI Reference Sequence: NM_173867 ; Ensembl: ENSMUSG00000040945 ) is located on Mouse chromosome 4. 13 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000038893). Exon 3~10 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note:
Exon 3 starts from about 17.95% of the coding region. Exon 3~10 covers 65.9% of the coding region. The size of effective KO region: ~10580 bp.
Page 1 of 8 https://www.alphaknockout.com
Overview of the Targeting Strategy
Wildtype allele 5' gRNA region gRNA region 3'
1 3 4 5 6 7 8 9 10 13
Legends Exon of mouse Rcc2 Knockout region
Page 2 of 8 https://www.alphaknockout.com
Overview of the Dot Plot (up) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 2000 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 609 bp section downstream of Exon 10 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.
Page 3 of 8 https://www.alphaknockout.com
Overview of the GC Content Distribution (up) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(21.95% 439) | C(23.75% 475) | T(24.75% 495) | G(29.55% 591)
Note: The 2000 bp section upstream of Exon 3 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.
Overview of the GC Content Distribution (down) Window size: 300 bp
Sequence 12
Summary: Full Length(609bp) | A(21.02% 128) | C(22.5% 137) | T(25.94% 158) | G(30.54% 186)
Note: The 609 bp section downstream of Exon 10 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 4 of 8 https://www.alphaknockout.com
BLAT Search Results (up)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr4 + 140705952 140707951 2000 browser details YourSeq 52 601 724 2000 87.2% chr11 + 115194341 115194465 125 browser details YourSeq 50 604 729 2000 93.2% chr12 - 103039933 103040059 127 browser details YourSeq 50 592 669 2000 88.9% chr11 - 60350408 60350701 294 browser details YourSeq 50 687 790 2000 93.2% chr15 + 100826696 100826858 163 browser details YourSeq 47 602 724 2000 92.9% chr18 + 36008721 36008844 124 browser details YourSeq 46 578 645 2000 84.9% chr12 + 29033871 29033941 71 browser details YourSeq 45 602 669 2000 82.2% chr7 + 102069331 102069394 64 browser details YourSeq 41 713 922 2000 93.4% chr16 - 14281484 14281903 420 browser details YourSeq 41 635 724 2000 95.6% chr16 + 69100121 69100210 90 browser details YourSeq 40 602 722 2000 93.5% chr18 - 34507939 34508060 122 browser details YourSeq 40 551 647 2000 69.7% chr17 + 7737208 7737287 80 browser details YourSeq 40 602 669 2000 79.3% chr10 + 82721461 82721524 64 browser details YourSeq 38 553 678 2000 93.2% chr16 + 31208661 31208788 128 browser details YourSeq 37 602 702 2000 95.3% chr5 - 144351861 144351961 101 browser details YourSeq 35 602 729 2000 80.0% chr14 - 55684134 55684256 123 browser details YourSeq 35 892 930 2000 94.9% chr5 + 114195927 114195965 39 browser details YourSeq 35 623 729 2000 90.7% chr2 + 80574821 80574929 109 browser details YourSeq 33 602 645 2000 88.4% chr2 - 122288457 122288501 45 browser details YourSeq 33 602 725 2000 94.6% chr17 - 46710191 46710314 124
Note: The 2000 bp section upstream of Exon 3 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 609 1 609 609 100.0% chr4 + 140717732 140718340 609 browser details YourSeq 29 24 77 609 65.7% chr3 - 102663698 102663731 34 browser details YourSeq 25 34 61 609 96.5% chr1 + 3690136 3690246 111 browser details YourSeq 23 39 62 609 100.0% chr10 - 121337598 121337626 29 browser details YourSeq 22 58 80 609 100.0% chr11 + 4996263 4996292 30 browser details YourSeq 21 389 409 609 100.0% chr1 + 194627345 194627365 21 browser details YourSeq 20 126 145 609 100.0% chr11 - 18452766 18452785 20 browser details YourSeq 20 562 585 609 91.7% chr11 + 5399623 5399646 24
Note: The 609 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found.
Page 5 of 8 https://www.alphaknockout.com
Gene and protein information: Rcc2 regulator of chromosome condensation 2 [ Mus musculus (house mouse) ] Gene ID: 108911, updated on 12-Aug-2019
Gene summary
Official Symbol Rcc2 provided by MGI Official Full Name regulator of chromosome condensation 2 provided by MGI Primary source MGI:MGI:1919784 See related Ensembl:ENSMUSG00000040945 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 Td60; AA536646; AA675016; mKIAA1470; 2610510H01Rik; 2610529N02Rik Expression Ubiquitous expression in thymus adult (RPKM 76.6), CNS E11.5 (RPKM 66.1) and 28 other tissues See more Orthologs human all
Genomic context
Location: 4; 4 D3 See Rcc2 in Genome Data Viewer Exon count: 14
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (140701473..140723220)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (140257388..140279135)
Chromosome 4 - NC_000070.6
Page 6 of 8 https://www.alphaknockout.com
Transcript information: This gene has 5 transcripts
Gene: Rcc2 ENSMUSG00000040945
Description regulator of chromosome condensation 2 [Source:MGI Symbol;Acc:MGI:1919784] Gene Synonyms 2610510H01Rik, 2610529N02Rik, Td60 Location Chromosome 4: 140,700,541-140,723,220 forward strand. GRCm38:CM000997.2 About this gene This gene has 5 transcripts (splice variants), 198 orthologues, 9 paralogues, is a member of 1 Ensembl protein family and is associated with 13 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Rcc2-202 ENSMUST00000071169.8 3999 520aa ENSMUSP00000071163.2 Protein coding CCDS18852 Q8BK67 TSL:1 GENCODE basic APPRIS P1
Rcc2-201 ENSMUST00000038893.5 3767 520aa ENSMUSP00000038144.5 Protein coding CCDS18852 Q8BK67 TSL:1 GENCODE basic APPRIS P1
Rcc2-205 ENSMUST00000138808.7 699 170aa ENSMUSP00000117448.1 Protein coding - A2AWQ2 CDS 3' incomplete TSL:2
Rcc2-204 ENSMUST00000138682.7 430 No protein - lncRNA - - TSL:2
Rcc2-203 ENSMUST00000129838.1 347 No protein - lncRNA - - TSL:5
42.68 kb Forward strand 140.70Mb 140.71Mb 140.72Mb 140.73Mb Genes (Comprehensive set... Gm23045-201 >miRNA Rcc2-202 >protein coding
Rcc2-201 >protein coding
Rcc2-205 >protein coding Rcc2-204 >lncRNA
Rcc2-203 >lncRNA
Gm25951-201 >miRNA
Contigs AL954710.27 >
Genes < Padi6-201protein coding (Comprehensive set...
< Padi6-202lncRNA
Regulatory Build
140.70Mb 140.71Mb 140.72Mb 140.73Mb Reverse strand 42.68 kb
Regulation Legend CTCF Enhancer Promoter Promoter Flank
Gene Legend Protein Coding
Ensembl protein coding merged Ensembl/Havana
Non-Protein Coding
RNA gene
Page 7 of 8 https://www.alphaknockout.com
Transcript: ENSMUST00000038893
21.75 kb Forward strand
Rcc2-201 >protein coding
ENSMUSP00000038... MobiDB lite Low complexity (Seg) Superfamily Regulator of chromosome condensation 1/beta-lactamase-inhibitor protein II Prints Regulator of chromosome condensation, RCC1 Pfam Regulator of chromosome condensation, RCC1 PROSITE profiles Regulator of chromosome condensation, RCC1 PROSITE patterns Regulator of chromosome condensation, RCC1 PANTHER PTHR46207 Gene3D Regulator of chromosome condensation 1/beta-lactamase-inhibitor protein II
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant splice region variant synonymous variant
Scale bar 0 60 120 180 240 300 360 420 520
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
Page 8 of 8