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Mouse Cul2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cul2 conditional 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 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cul2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-192L4 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 4 starts from about 9.98% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 8342 bp, and the size of intron 4 for 3'-loxP site insertion: 3310 bp. The size of effective cKO region: ~595 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 4 21 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cul2 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(7095bp) | A(29.09% 2064) | C(18.96% 1345) | T(30.8% 2185) | G(21.16% 1501)

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% chr18 + 3410881 3413880 3000 browser details YourSeq 1027 1 1209 3000 93.1% chr4 + 117944229 117945512 1284 browser details YourSeq 1019 1 1221 3000 92.6% chr4 - 11908553 11909833 1281 browser details YourSeq 1013 1 1210 3000 92.5% chr16 - 11722129 11723405 1277 browser details YourSeq 1012 9 1210 3000 92.5% chr19 + 15382977 15384244 1268 browser details YourSeq 1011 1 1208 3000 92.9% chr12 - 119252489 119253760 1272 browser details YourSeq 1009 1 1220 3000 92.1% chr8 - 110646880 110648160 1281 browser details YourSeq 1009 1 1209 3000 92.9% chr5 - 130486442 130487707 1266 browser details YourSeq 1009 1 1209 3000 92.4% chrX + 10669561 10670838 1278 browser details YourSeq 1009 1 1209 3000 92.6% chr10 + 8670402 8671684 1283 browser details YourSeq 1008 1 1209 3000 92.3% chr2 + 139889502 139890779 1278 browser details YourSeq 1008 1 1219 3000 92.0% chr17 + 74130216 74131496 1281 browser details YourSeq 1007 1 1220 3000 93.0% chr10 - 58266192 58267675 1484 browser details YourSeq 1006 1 1209 3000 93.1% chr13 - 59222455 59223720 1266 browser details YourSeq 1005 1 1211 3000 92.0% chr8 - 64809089 64810365 1277 browser details YourSeq 1005 1 1207 3000 92.5% chr11 + 115576391 115577662 1272 browser details YourSeq 1004 1 1211 3000 92.2% chr12 - 5468419 5469694 1276 browser details YourSeq 1004 1 1211 3000 92.4% chr3 + 102642613 102643889 1277 browser details YourSeq 1002 1 1220 3000 92.2% chr18 + 12327823 12329075 1253 browser details YourSeq 1001 1 1213 3000 92.2% chr11 - 20942050 20943339 1290

Note: The 3000 bp section upstream of Exon 4 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% chr18 + 3414476 3417475 3000 browser details YourSeq 92 1937 2218 3000 86.9% chr11 + 69748729 69749244 516 browser details YourSeq 71 1937 2086 3000 93.9% chr10 - 95484596 95484755 160 browser details YourSeq 71 1978 2079 3000 87.4% chr11 + 98957624 98957749 126 browser details YourSeq 70 1952 2086 3000 91.6% chr13 - 50967211 50967361 151 browser details YourSeq 70 1940 2086 3000 91.7% chr1 + 8022555 8022721 167 browser details YourSeq 69 1994 2084 3000 92.6% chr17 - 28223142 28223244 103 browser details YourSeq 69 1991 2083 3000 89.7% chr13 + 48644666 48644784 119 browser details YourSeq 69 1941 2086 3000 93.7% chr12 + 61221865 61222026 162 browser details YourSeq 68 1584 2086 3000 76.0% chr8 - 84221835 84222244 410 browser details YourSeq 68 1953 2081 3000 92.5% chr1 - 73875132 73875271 140 browser details YourSeq 66 1940 2086 3000 95.9% chr1 - 68738030 68738179 150 browser details YourSeq 66 1941 2047 3000 79.6% chr7 + 127639971 127640060 90 browser details YourSeq 66 1991 2084 3000 87.5% chr6 + 99444757 99444876 120 browser details YourSeq 65 1987 2080 3000 87.5% chr10 - 4104184 4104301 118 browser details YourSeq 65 1978 2218 3000 95.9% chr14 + 71054805 71055332 528 browser details YourSeq 64 1978 2079 3000 91.2% chr11 - 86528819 86529098 280 browser details YourSeq 64 1953 2079 3000 91.0% chr12 + 22065909 22066054 146 browser details YourSeq 61 1965 2083 3000 92.9% chr11 - 34828320 34828462 143 browser details YourSeq 60 1997 2086 3000 90.7% chr1 - 193344522 193344646 125

Note: The 3000 bp section downstream of Exon 4 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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