https://www.alphaknockout.com

Mouse Cul2 Knockout Project (CRISPR/Cas9)

Objective: To create a Cul2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cul2 gene (NCBI Reference Sequence: NM_029402 ; Ensembl: ENSMUSG00000024231 ) is located on Mouse chromosome 18. 21 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 21 (Transcript: ENSMUST00000162301). Exon 5~16 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 5 starts from about 14.23% of the coding region. Exon 5~16 covers 58.17% of the coding region. The size of effective KO region: ~9706 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3' 14

1 5 6 7 8 9 10 11 12 13 15 16 21

Legends Exon of mouse Cul2 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 5 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 2000 bp section downstream of Exon 16 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(23.9% 478) | C(18.5% 370) | T(35.8% 716) | G(21.8% 436)

Note: The 2000 bp section upstream of Exon 5 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(2000bp) | A(28.4% 568) | C(15.0% 300) | T(32.75% 655) | G(23.85% 477)

Note: The 2000 bp section downstream of Exon 16 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 2000 1 2000 2000 100.0% chr18 + 3415536 3417535 2000 browser details YourSeq 92 877 1158 2000 86.9% chr11 + 69748729 69749244 516 browser details YourSeq 71 877 1026 2000 93.9% chr10 - 95484596 95484755 160 browser details YourSeq 71 935 1175 2000 88.9% chr13 + 113867158 113867421 264 browser details YourSeq 71 918 1019 2000 87.4% chr11 + 98957624 98957749 126 browser details YourSeq 70 892 1026 2000 91.6% chr13 - 50967211 50967361 151 browser details YourSeq 70 880 1026 2000 91.7% chr1 + 8022555 8022721 167 browser details YourSeq 69 934 1024 2000 92.6% chr17 - 28223142 28223244 103 browser details YourSeq 69 931 1023 2000 89.7% chr13 + 48644666 48644784 119 browser details YourSeq 69 881 1026 2000 93.7% chr12 + 61221865 61222026 162 browser details YourSeq 68 524 1026 2000 76.0% chr8 - 84221835 84222244 410 browser details YourSeq 68 893 1021 2000 92.5% chr1 - 73875132 73875271 140 browser details YourSeq 66 880 1026 2000 95.9% chr1 - 68738030 68738179 150 browser details YourSeq 66 881 987 2000 79.6% chr7 + 127639971 127640060 90 browser details YourSeq 66 931 1024 2000 87.5% chr6 + 99444757 99444876 120 browser details YourSeq 65 918 1158 2000 95.9% chr14 + 71054805 71055332 528 browser details YourSeq 64 918 1019 2000 91.2% chr11 - 86528819 86529098 280 browser details YourSeq 64 893 1019 2000 91.0% chr12 + 22065909 22066054 146 browser details YourSeq 63 915 1019 2000 88.9% chr15 - 55026521 55026652 132 browser details YourSeq 61 905 1023 2000 92.9% chr11 - 34828320 34828462 143

Note: The 2000 bp section upstream of Exon 5 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 2000 1 2000 2000 100.0% chr18 + 3427242 3429241 2000 browser details YourSeq 189 41 280 2000 94.6% chr3 - 149058657 149059312 656 browser details YourSeq 187 41 278 2000 95.0% chr8 + 8933487 8933971 485 browser details YourSeq 181 41 280 2000 93.8% chr3 - 149059050 149059386 337 browser details YourSeq 176 50 278 2000 93.7% chr8 + 8933565 8934015 451 browser details YourSeq 176 48 278 2000 97.0% chr11 + 6699688 6884809 185122 browser details YourSeq 175 41 278 2000 94.5% chr6 - 11340941 11341188 248 browser details YourSeq 170 50 280 2000 94.9% chr3 - 149058588 149059287 700 browser details YourSeq 165 50 278 2000 90.2% chr11 + 16558506 16558710 205 browser details YourSeq 161 50 278 2000 95.7% chr8 + 8933341 8933995 655 browser details YourSeq 160 50 278 2000 96.6% chr6 - 11340709 11341064 356 browser details YourSeq 155 54 280 2000 95.5% chr3 - 149058981 149059419 439 browser details YourSeq 152 62 280 2000 94.9% chr3 - 149059009 149059436 428 browser details YourSeq 152 51 278 2000 93.3% chr1 + 93682902 93683191 290 browser details YourSeq 151 50 278 2000 97.0% chr6 - 11340865 11341102 238 browser details YourSeq 139 50 229 2000 93.1% chr6 - 11340616 11340817 202 browser details YourSeq 133 66 278 2000 93.7% chr11 + 6699709 6700101 393 browser details YourSeq 132 100 278 2000 96.6% chr8 - 116617012 116617282 271 browser details YourSeq 130 50 223 2000 88.8% chr18 - 78985330 78985481 152 browser details YourSeq 125 104 274 2000 94.6% chr15 - 99538778 99539061 284

Note: The 2000 bp section downstream of Exon 16 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cul2 cullin 2 [ Mus musculus (house mouse) ] Gene ID: 71745, updated on 24-Oct-2019

Gene summary

Official Symbol Cul2 provided by MGI Official Full Name cullin 2 provided by MGI Primary source MGI:MGI:1918995 See related Ensembl:ENSMUSG00000024231 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 AI327301; mKIAA4106; 1300003D18Rik; 4932411N15Rik Expression Ubiquitous expression in placenta adult (RPKM 8.3), CNS E18 (RPKM 7.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 18; 18 A1 See Cul2 in Genome Data Viewer Exon count: 23

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (3382970..3436700)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (3383223..3436698)

Chromosome 18 - NC_000084.6

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Transcript information: This gene has 7 transcripts

Gene: Cul2 ENSMUSG00000024231

Description cullin 2 [Source:MGI Symbol;Acc:MGI:1918995] Gene Synonyms 1300003D18Rik, 4932411N15Rik Location Chromosome 18: 3,382,988-3,436,377 forward strand. GRCm38:CM001011.2 About this gene This gene has 7 transcripts (splice variants), 211 orthologues, 7 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cul2- ENSMUST00000162301.7 3828 745aa ENSMUSP00000125403.1 Protein coding CCDS29032 Q9D4H8 TSL:1 205 GENCODE basic APPRIS P1

Cul2- ENSMUST00000025073.11 3727 706aa ENSMUSP00000025073.5 Protein coding - Q9D4H8 TSL:1 201 GENCODE basic

Cul2- ENSMUST00000161317.1 1909 582aa ENSMUSP00000123903.1 Protein coding - E0CYT5 CDS 3' 204 incomplete TSL:5

Cul2- ENSMUST00000080089.10 2799 90aa ENSMUSP00000078988.4 Nonsense mediated - H7BX52 TSL:1 202 decay

Cul2- ENSMUST00000234843.1 993 49aa ENSMUSP00000157267.1 Nonsense mediated - A0A3Q4EI83 - 207 decay

Cul2- ENSMUST00000234414.1 618 111aa ENSMUSP00000157136.1 Nonsense mediated - A0A3Q4EBZ1 - 206 decay

Cul2- ENSMUST00000159443.1 397 No - Retained intron - - TSL:2 203 protein

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73.39 kb Forward strand 3.38Mb 3.40Mb 3.42Mb 3.44Mb Genes (Comprehensive set... Cul2-201 >protein coding

Cul2-202 >nonsense mediated decay

Cul2-205 >protein coding

Cul2-207 >nonsense mediated decay Cul2-203 >retained intron

Cul2-204 >protein coding

Cul2-206 >nonsense mediated decay

Contigs < AC124336.10 Regulatory Build

3.38Mb 3.40Mb 3.42Mb 3.44Mb Reverse strand 73.39 kb

Regulation Legend

Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000162301

53.15 kb Forward strand

Cul2-205 >protein coding

ENSMUSP00000125... Low complexity (Seg) Superfamily Cullin repeat-like-containing domain superfamily Cullin homology domain superfamily Winged helix DNA-binding domain superfamily

SMART Cullin homology domain Cullin protein, neddylation domain

Pfam Cullin, N-terminal Cullin protein, neddylation domain

PROSITE profiles Cullin homology domain PROSITE patterns Cullin, conserved site PANTHER PTHR11932

PTHR11932:SF131 Gene3D 1.20.1310.10 1.10.10.2620 Winged helix-like DNA-binding domain superfamily

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 745

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

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