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Mouse Cdc27 Knockout Project (CRISPR/Cas9)

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Mouse Cdc27 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Cdc27 Knockout Project (CRISPR/Cas9) Objective: To create a Cdc27 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cdc27 gene (NCBI Reference Sequence: NM_145436 ; Ensembl: ENSMUSG00000020687 ) is located on Mouse chromosome 11. 19 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 19 (Transcript: ENSMUST00000093923). Exon 2~7 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 2 starts from about 1.13% of the coding region. Exon 2~7 covers 32.93% of the coding region. The size of effective KO region: ~8408 bp. The KO 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 2 3 4 5 6 7 19 Legends Exon of mouse Cdc27 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 2 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. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1285 bp section downstream of Exon 7 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(32.9% 658) | C(18.65% 373) | T(26.15% 523) | G(22.3% 446) Note: The 2000 bp section upstream of Exon 2 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(1285bp) | A(31.75% 408) | C(17.67% 227) | T(30.66% 394) | G(19.92% 256) Note: The 1285 bp section downstream of Exon 7 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% chr11 - 104536671 104538670 2000 browser details YourSeq 145 1265 1424 2000 95.7% chr9 - 48335362 48335522 161 browser details YourSeq 134 1270 1423 2000 92.8% chr15 - 51932842 51932994 153 browser details YourSeq 134 1266 1424 2000 93.0% chr11 - 94380358 94380521 164 browser details YourSeq 132 1188 1403 2000 92.4% chr16 + 90251244 90251790 547 browser details YourSeq 131 1265 1427 2000 91.3% chr10 - 80815366 80815554 189 browser details YourSeq 131 1274 1422 2000 94.0% chr15 + 99808925 99809073 149 browser details YourSeq 130 1266 1418 2000 93.4% chr17 + 78676201 78676353 153 browser details YourSeq 128 1265 1419 2000 89.0% chr14 + 46578863 46579008 146 browser details YourSeq 127 1262 1421 2000 93.2% chr4 - 140734650 140734809 160 browser details YourSeq 127 1266 1425 2000 94.5% chr18 + 32811079 32811251 173 browser details YourSeq 127 1274 1418 2000 94.5% chr1 + 144801360 144801507 148 browser details YourSeq 126 1263 1405 2000 94.4% chr14 - 122040294 122040437 144 browser details YourSeq 125 1265 1413 2000 91.2% chr18 - 19115095 19115242 148 browser details YourSeq 125 1265 1423 2000 90.8% chr1 - 36314044 36314209 166 browser details YourSeq 125 7 167 2000 93.3% chr2 + 154753353 154777827 24475 browser details YourSeq 125 1259 1412 2000 91.5% chr10 + 4666879 4667039 161 browser details YourSeq 124 1262 1424 2000 89.3% chr2 - 132068021 132068181 161 browser details YourSeq 124 1263 1419 2000 86.1% chr17 - 59193928 59194078 151 browser details YourSeq 124 1265 1424 2000 88.0% chr16 - 84190134 84190292 159 Note: The 2000 bp section upstream of Exon 2 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 1285 1 1285 1285 100.0% chr11 - 104526978 104528262 1285 browser details YourSeq 138 300 625 1285 86.7% chr10 + 84982458 84982729 272 browser details YourSeq 134 472 635 1285 94.7% chrX - 13299099 13299262 164 browser details YourSeq 132 480 625 1285 96.6% chr15 - 77676827 77676972 146 browser details YourSeq 130 255 625 1285 83.5% chr11 - 89939083 89939387 305 browser details YourSeq 130 460 629 1285 87.1% chr11 + 111103698 111103855 158 browser details YourSeq 127 475 625 1285 93.9% chr7 - 34628974 34629126 153 browser details YourSeq 127 479 629 1285 95.1% chr10 - 82830831 82830981 151 browser details YourSeq 126 478 629 1285 92.7% chr11 + 70401206 70401357 152 browser details YourSeq 125 477 625 1285 94.4% chr13 - 77711623 77711772 150 browser details YourSeq 124 479 625 1285 95.0% chr7 - 3273605 3273751 147 browser details YourSeq 124 488 650 1285 87.8% chr3 - 62422520 62422671 152 browser details YourSeq 124 472 625 1285 93.4% chr19 - 5590354 5590505 152 browser details YourSeq 124 479 625 1285 93.2% chr1 - 192342156 192342303 148 browser details YourSeq 124 480 625 1285 93.8% chr3 + 88275740 88275885 146 browser details YourSeq 124 478 625 1285 93.2% chr16 + 13854201 13854348 148 browser details YourSeq 124 480 625 1285 93.8% chr16 + 11662643 11662788 146 browser details YourSeq 123 479 616 1285 96.3% chr18 - 70443919 70444056 138 browser details YourSeq 123 483 629 1285 95.0% chr17 - 56640470 56640646 177 browser details YourSeq 123 477 625 1285 94.9% chr10 - 62769819 62769967 149 Note: The 1285 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Cdc27 cell division cycle 27 [ Mus musculus (house mouse) ] Gene ID: 217232, updated on 12-Aug-2019 Gene summary Official Symbol Cdc27 provided by MGI Official Full Name cell division cycle 27 provided by MGI Primary source MGI:MGI:102685 See related Ensembl:ENSMUSG00000020687 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 APC3; AI452358; BC023187 Expression Ubiquitous expression in CNS E11.5 (RPKM 13.7), placenta adult (RPKM 12.0) and 25 other tissues See more Orthologs human all Genomic context Location: 11 E1; 11 67.79 cM See Cdc27 in Genome Data Viewer Exon count: 19 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (104502524..104550620, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (104363838..104411934, complement) Chromosome 11 - NC_000077.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Cdc27 ENSMUSG00000020687 Description cell division cycle 27 [Source:MGI Symbol;Acc:MGI:102685] Gene Synonyms APC3 Location Chromosome 11: 104,502,745-104,550,620 reverse strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 207 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cdc27-201 ENSMUST00000093923.8 5425 825aa ENSMUSP00000091452.2 Protein coding CCDS36354 A2A6Q5 TSL:1 GENCODE basic APPRIS P2 Cdc27-203 ENSMUST00000106962.8 3160 831aa ENSMUSP00000102575.2 Protein coding - A2A6Q5 TSL:5 GENCODE basic APPRIS ALT1 Cdc27-202 ENSMUST00000106961.1 1863 399aa ENSMUSP00000102574.1 Protein coding - Q8R568 TSL:1 GENCODE basic Cdc27-204 ENSMUST00000127506.1 580 No protein - lncRNA - - TSL:3 Cdc27-205 ENSMUST00000135303.1 486 No protein - lncRNA - - TSL:2 67.88 kb Forward strand 104.50Mb 104.52Mb 104.54Mb 104.56Mb Genes Myl4-201 >protein coding (Comprehensive set... Contigs AL603709.14 > Genes (Comprehensive set... < Cdc27-201protein coding < Gm22000-201lncRNA < Cdc27-203protein coding < Gm11665-201lncRNA < Cdc27-204lncRNA < Cdc27-202protein coding < Cdc27-205lncRNA Regulatory Build 104.50Mb 104.52Mb 104.54Mb 104.56Mb Reverse strand 67.88 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000093923 < Cdc27-201protein coding Reverse strand 47.74 kb ENSMUSP00000091... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Tetratricopeptide-like helical domain superfamily SMART Tetratricopeptide repeat Pfam PF12895 Tetratricopeptide repeat 1 Tetratricopeptide repeat PROSITE profiles Tetratricopeptide repeat Tetratricopeptide repeat-containing domain PANTHER PTHR12558:SF13 PTHR12558 Gene3D Tetratricopeptide-like helical domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 825 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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