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Mouse Cul1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Cul1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Cul1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Cul1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cul1 gene (NCBI Reference Sequence: NM_012042 ; Ensembl: ENSMUSG00000029686 ) is located on Mouse chromosome 6. 22 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 22 (Transcript: ENSMUST00000031697). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cul1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-18I21 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: Homozygotes for targeted null mutations accumulate cyclin E1 and exhibit arrested development and lethality around embryonic day 6.5. Exon 5 starts from about 20.79% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 3779 bp, and the size of intron 6 for 3'-loxP site insertion: 637 bp. The size of effective cKO region: ~1122 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 5 6 7 22 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Cul1 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. 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(7622bp) | A(30.19% 2301) | C(17.32% 1320) | T(33.1% 2523) | G(19.39% 1478) 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. 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% chr6 + 47497919 47500918 3000 browser details YourSeq 67 742 1252 3000 71.5% chr1 + 13298294 13298637 344 browser details YourSeq 49 1178 1261 3000 78.0% chr14 - 54727108 54727186 79 browser details YourSeq 48 234 401 3000 92.9% chr5 + 9567456 9567645 190 browser details YourSeq 47 742 802 3000 90.0% chr11 - 80326756 80327231 476 browser details YourSeq 43 742 803 3000 93.8% chr11 + 33184609 33184827 219 browser details YourSeq 38 347 411 3000 93.2% chr1 + 183349635 183349699 65 browser details YourSeq 37 2107 2143 3000 100.0% chr6 + 47500917 47500953 37 browser details YourSeq 37 1550 1591 3000 95.3% chr11 + 116633751 116633793 43 browser details YourSeq 36 593 769 3000 92.7% chr1 - 29808005 29808181 177 browser details YourSeq 35 738 802 3000 94.9% chr1 - 97667269 97667351 83 browser details YourSeq 34 785 828 3000 90.5% chrX - 17705226 17705277 52 browser details YourSeq 34 1212 1253 3000 97.3% chr11 - 50264445 50264775 331 browser details YourSeq 32 2051 2108 3000 92.2% chr1 - 138882566 138882624 59 browser details YourSeq 32 2063 2103 3000 92.2% chr10 + 115413275 115413316 42 browser details YourSeq 31 2063 2110 3000 91.9% chr11 + 22920365 22920413 49 browser details YourSeq 30 2063 2108 3000 94.3% chr14 - 26085621 26085666 46 browser details YourSeq 30 2650 2689 3000 87.5% chr1 - 188269788 188269827 40 browser details YourSeq 29 231 262 3000 96.9% chr1 - 119299669 119299701 33 browser details YourSeq 29 2064 2103 3000 96.8% chr15 + 100396715 100396755 41 Note: The 3000 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 3000 1 3000 3000 100.0% chr6 + 47502041 47505040 3000 browser details YourSeq 262 1383 2843 3000 95.0% chr5 + 140409332 140466292 56961 browser details YourSeq 162 1315 1525 3000 89.7% chr4 + 141250162 141250356 195 browser details YourSeq 153 2669 2844 3000 93.5% chr7 - 141046193 141046366 174 browser details YourSeq 147 1377 1677 3000 93.5% chr19 + 16158279 16158819 541 browser details YourSeq 147 2673 2850 3000 92.0% chr13 + 106203573 106203745 173 browser details YourSeq 143 2674 2848 3000 91.0% chr8 + 104578499 104578671 173 browser details YourSeq 143 1383 2022 3000 83.3% chr8 + 83970416 83970871 456 browser details YourSeq 143 2682 2887 3000 94.0% chr5 + 15750277 15750763 487 browser details YourSeq 141 2682 2846 3000 91.3% chrX + 46577450 46577611 162 browser details YourSeq 140 1366 1526 3000 91.9% chr17 - 34207242 34207400 159 browser details YourSeq 140 1333 1525 3000 91.7% chr3 + 137988589 137988807 219 browser details YourSeq 140 1321 1520 3000 92.2% chr10 + 102119521 102119968 448 browser details YourSeq 139 2678 2844 3000 90.3% chr11 + 6542533 6542690 158 browser details YourSeq 137 1366 1521 3000 92.3% chr5 - 23469607 23469760 154 browser details YourSeq 136 1337 1526 3000 94.8% chr18 - 74949506 74949921 416 browser details YourSeq 136 1366 1526 3000 89.9% chr16 - 96006818 96006975 158 browser details YourSeq 136 2673 2842 3000 89.5% chr19 + 53744237 53744392 156 browser details YourSeq 136 1362 1523 3000 93.7% chr1 + 26022446 26022622 177 browser details YourSeq 135 1362 1525 3000 88.9% chr12 + 97270093 97270244 152 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Cul1 cullin 1 [ Mus musculus (house mouse) ] Gene ID: 26965, updated on 24-Oct-2019 Gene summary Official Symbol Cul1 provided by MGI Official Full Name cullin 1 provided by MGI Primary source MGI:MGI:1349658 See related Ensembl:ENSMUSG00000029686 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 Cul-1 Expression Ubiquitous expression in CNS E11.5 (RPKM 25.9), bladder adult (RPKM 24.8) and 28 other tissues See more Orthologs human all Genomic context Location: 6; 6 B2.3 See Cul1 in Genome Data Viewer Exon count: 25 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (47453732..47526144) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (47404323..47476138) Chromosome 6 - NC_000072.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Cul1 ENSMUSG00000029686 Description cullin 1 [Source:MGI Symbol;Acc:MGI:1349658] Location Chromosome 6: 47,453,398-47,526,139 forward strand. GRCm38:CM000999.2 About this gene This gene has 9 transcripts (splice variants), 230 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cul1-201 ENSMUST00000031697.8 3161 776aa ENSMUSP00000031697.8 Protein coding CCDS20095 Q9WTX6 TSL:1 GENCODE basic APPRIS P1 Cul1-204 ENSMUST00000146200.7 2385 699aa ENSMUSP00000122702.1 Protein coding - Q3TPM3 CDS 3' incomplete TSL:1 Cul1-205 ENSMUST00000146828.7 723 110aa ENSMUSP00000121608.1 Protein coding - D3Z2H3 CDS 3' incomplete TSL:2 Cul1-203 ENSMUST00000143941.2 530 51aa ENSMUSP00000144845.1 Protein coding - A0A0N4SUW1 TSL:1 GENCODE basic Cul1-208 ENSMUST00000152547.5 501 20aa ENSMUSP00000114959.1 Protein coding - D3YUS8 CDS 3' incomplete TSL:2 Cul1-207 ENSMUST00000151934.1 569 No protein - Retained intron - - TSL:5 Cul1-209 ENSMUST00000154201.1 381 No protein - Retained intron - - TSL:3 Cul1-206 ENSMUST00000149531.1 581 No protein - lncRNA - - TSL:2 Cul1-202 ENSMUST00000126877.1 368 No protein - lncRNA - - TSL:3 Page 6 of 8 https://www.alphaknockout.com 92.74 kb Forward strand 47.46Mb 47.48Mb 47.50Mb 47.52Mb Genes (Comprehensive set... Cul1-205 >protein coding Cul1-202 >lncRNA Cul1-208 >protein coding Cul1-207 >retained intron Cul1-204 >protein coding Cul1-201 >protein coding Cul1-203 >protein coding Cul1-209 >retained intron Cul1-206 >lncRNA Contigs < AC154014.4 < AC154015.3 Genes < A930035D04Rik-201pseudogene < Ezh2-213retained intron (Comprehensive set... < Ezh2-212retained intron < Ezh2-210retained intron < Ezh2-207retained intron < Ezh2-203protein coding < Ezh2-204protein coding < Ezh2-201protein coding < Ezh2-214protein coding < Ezh2-202protein coding Regulatory Build 47.46Mb 47.48Mb 47.50Mb 47.52Mb Reverse strand 92.74 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000031697 71.82 kb Forward strand Cul1-201 >protein coding ENSMUSP00000031... Low complexity (Seg) Coiled-coils (Ncoils) 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:SF81 Gene3D 1.20.1310.10 4.10.1030.10 Winged helix-like DNA-binding domain superfamily All sequence SNPs/i..
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