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Mouse Rcc2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rcc2 conditional 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 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rcc2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-275J10 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 3 starts from about 17.95% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 5515 bp, and the size of intron 3 for 3'-loxP site insertion: 2288 bp. The size of effective cKO region: ~594 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rcc2 Homology arm cKO region loxP site

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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(7094bp) | A(19.82% 1406) | C(24.44% 1734) | T(25.95% 1841) | G(29.79% 2113)

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.

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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 + 140704702 140707701 3000 browser details YourSeq 52 1851 1974 3000 87.2% chr11 + 115194341 115194465 125 browser details YourSeq 50 1854 1979 3000 93.2% chr12 - 103039933 103040059 127 browser details YourSeq 50 1842 1919 3000 88.9% chr11 - 60350408 60350701 294 browser details YourSeq 50 1937 2040 3000 93.2% chr15 + 100826696 100826858 163 browser details YourSeq 47 1852 1974 3000 92.9% chr18 + 36008721 36008844 124 browser details YourSeq 46 1828 1895 3000 84.9% chr12 + 29033871 29033941 71 browser details YourSeq 45 1852 1919 3000 82.2% chr7 + 102069331 102069394 64 browser details YourSeq 41 1963 2172 3000 93.4% chr16 - 14281484 14281903 420 browser details YourSeq 41 1885 1974 3000 95.6% chr16 + 69100121 69100210 90 browser details YourSeq 40 1852 1972 3000 93.5% chr18 - 34507939 34508060 122 browser details YourSeq 40 1801 1897 3000 69.7% chr17 + 7737208 7737287 80 browser details YourSeq 40 1852 1919 3000 79.3% chr10 + 82721461 82721524 64 browser details YourSeq 38 1803 1928 3000 93.2% chr16 + 31208661 31208788 128 browser details YourSeq 37 1852 1952 3000 95.3% chr5 - 144351861 144351961 101 browser details YourSeq 35 1852 1979 3000 80.0% chr14 - 55684134 55684256 123 browser details YourSeq 35 2142 2180 3000 94.9% chr5 + 114195927 114195965 39 browser details YourSeq 35 1873 1979 3000 90.7% chr2 + 80574821 80574929 109 browser details YourSeq 33 1852 1975 3000 94.6% chr17 - 46710191 46710314 124 browser details YourSeq 31 1852 1895 3000 86.4% chr13 + 107835796 107835840 45

Note: The 3000 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 3000 1 3000 3000 100.0% chr4 + 140708296 140711295 3000 browser details YourSeq 30 2135 2166 3000 100.0% chr1 + 38238301 38238500 200 browser details YourSeq 22 2956 2978 3000 100.0% chr5 + 3680364 3680388 25 browser details YourSeq 22 698 719 3000 100.0% chr16 + 58610070 58610091 22 browser details YourSeq 22 193 218 3000 92.4% chr16 + 11537426 11537451 26 browser details YourSeq 21 2743 2763 3000 100.0% chr16 + 27471731 27471751 21 browser details YourSeq 21 613 634 3000 100.0% chr1 + 42484681 42484703 23 browser details YourSeq 20 2000 2021 3000 95.5% chr1 - 76264750 76264771 22 browser details YourSeq 20 947 966 3000 100.0% chr1 - 29867079 29867098 20

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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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

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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

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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.

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