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Mouse Cpsf6 Knockout Project (CRISPR/Cas9)

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Mouse Cpsf6 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Cpsf6 Knockout Project (CRISPR/Cas9) Objective: To create a Cpsf6 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cpsf6 gene (NCBI Reference Sequence: NM_001013391 ; Ensembl: ENSMUSG00000055531 ) is located on Mouse chromosome 10. 10 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000069168). Exon 2~8 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 3.69% of the coding region. Exon 2~8 covers 85.24% of the coding region. The size of effective KO region: ~8996 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 8 10 Legends Exon of mouse Cpsf6 Knockout region Page 2 of 9 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 2000 bp section downstream of Exon 8 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(30.55% 611) | C(16.75% 335) | T(34.65% 693) | G(18.05% 361) 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(2000bp) | A(26.95% 539) | C(19.15% 383) | T(34.9% 698) | G(19.0% 380) Note: The 2000 bp section downstream of Exon 8 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 9 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% chr10 - 117367989 117369988 2000 browser details YourSeq 150 1233 1437 2000 89.5% chr1 - 131449744 131449956 213 browser details YourSeq 150 1248 1435 2000 90.4% chr4 + 132910025 132958395 48371 browser details YourSeq 149 1235 1439 2000 86.7% chr11 - 7726768 7727015 248 browser details YourSeq 149 1249 1435 2000 88.8% chr11 + 117699633 117699818 186 browser details YourSeq 148 1246 1435 2000 87.9% chr4 + 137327186 137327374 189 browser details YourSeq 148 1104 1435 2000 79.9% chr11 + 106931156 106931350 195 browser details YourSeq 148 1232 1437 2000 87.8% chr1 + 179219156 179219364 209 browser details YourSeq 147 1247 1433 2000 88.2% chr2 - 26804259 26804444 186 browser details YourSeq 147 1231 1435 2000 85.8% chr1 - 86539753 86539955 203 browser details YourSeq 146 1248 1435 2000 89.3% chr2 + 153351896 153352084 189 browser details YourSeq 145 1249 1435 2000 89.7% chr16 - 17188161 17188580 420 browser details YourSeq 144 1241 1435 2000 87.5% chr18 + 60737725 60737919 195 browser details YourSeq 143 1231 1425 2000 87.4% chr5 - 36757872 36758067 196 browser details YourSeq 143 1249 1435 2000 86.5% chr6 + 70852466 70852650 185 browser details YourSeq 142 1249 1435 2000 88.2% chr9 - 110308528 110308714 187 browser details YourSeq 142 1248 1435 2000 86.7% chr12 - 40319752 40319938 187 browser details YourSeq 142 1248 1435 2000 86.7% chr11 - 104260527 104260713 187 browser details YourSeq 141 1248 1435 2000 88.2% chr7 - 99585041 99585229 189 browser details YourSeq 141 1249 1436 2000 87.1% chr7 + 28927254 28927440 187 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 2000 1 2000 2000 100.0% chr10 - 117356993 117358992 2000 browser details YourSeq 139 881 1197 2000 80.6% chr1 - 171944416 171944666 251 browser details YourSeq 129 916 1191 2000 83.4% chr11 - 77990416 77990617 202 browser details YourSeq 126 1165 1852 2000 91.5% chr11 + 100869646 101257195 387550 browser details YourSeq 125 868 1036 2000 91.6% chr13 - 112135229 112135402 174 browser details YourSeq 125 856 1036 2000 87.1% chr10 - 115282191 115282369 179 browser details YourSeq 124 887 1452 2000 78.4% chr16 - 91903185 91903347 163 browser details YourSeq 122 1718 1873 2000 91.2% chr4 - 120292708 120292884 177 browser details YourSeq 121 1718 1882 2000 91.2% chr10 + 116372009 116372183 175 browser details YourSeq 116 1405 1870 2000 79.4% chr2 - 155757522 155757766 245 browser details YourSeq 116 1718 1871 2000 89.7% chr12 - 101900696 101900857 162 browser details YourSeq 116 881 1036 2000 86.5% chr5 + 123259423 123259574 152 browser details YourSeq 116 1718 1891 2000 89.3% chr11 + 52375676 52375864 189 browser details YourSeq 116 1165 1843 2000 92.0% chr1 + 184805686 184891276 85591 browser details YourSeq 115 1720 1861 2000 92.6% chr6 - 144916722 144916890 169 browser details YourSeq 115 868 1448 2000 76.0% chr4 - 117039170 117039341 172 browser details YourSeq 115 907 1047 2000 91.5% chr10 - 63634374 63634516 143 browser details YourSeq 115 1719 1871 2000 89.6% chr2 + 163110653 163110810 158 browser details YourSeq 114 881 1036 2000 86.2% chr14 + 61742278 61742428 151 browser details YourSeq 114 1722 1867 2000 91.4% chr14 + 34843442 34843601 160 Note: The 2000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Cpsf6 cleavage and polyadenylation specific factor 6 [ Mus musculus (house mouse) ] Gene ID: 432508, updated on 12-Aug-2019 Gene summary Official Symbol Cpsf6 provided by MGI Official Full Name cleavage and polyadenylation specific factor 6 provided by MGI Primary source MGI:MGI:1913948 See related Ensembl:ENSMUSG00000055531 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 CFIM; CFIM68; AI256641; HPBRII-4; HPBRII-7; 4733401N12Rik Expression Ubiquitous expression in whole brain E14.5 (RPKM 23.5), CNS E14 (RPKM 23.0) and 28 other tissues See more Orthologs human all Genomic context Location: 10; 10 D2 See Cpsf6 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (117344667..117376998, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (116781724..116814029, complement) Chromosome 10 - NC_000076.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Cpsf6 ENSMUSG00000055531 Description cleavage and polyadenylation specific factor 6 [Source:MGI Symbol;Acc:MGI:1913948] Gene Synonyms 4733401N12Rik, CFIM68, HPBRII-4, HPBRII-7 Location Chromosome 10: 117,344,673-117,380,015 reverse strand. GRCm38:CM001003.2 About this gene This gene has 8 transcripts (splice variants), 183 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cpsf6- ENSMUST00000069168.12 6526 551aa ENSMUSP00000068408.6 Protein coding CCDS24193 Q6NVF9 TSL:1 201 GENCODE basic APPRIS P3 Cpsf6- ENSMUST00000177145.7 3819 551aa ENSMUSP00000135136.1 Protein coding CCDS24193 Q6NVF9 TSL:1 208 GENCODE basic APPRIS P3 Cpsf6- ENSMUST00000176686.7 2200 552aa ENSMUSP00000134823.1 Protein coding CCDS78898 H3BJ30 TSL:1 207 GENCODE basic APPRIS ALT1 Cpsf6- ENSMUST00000176670.7 2227 588aa ENSMUSP00000135150.1 Protein coding - H3BJW3 TSL:5 206 GENCODE basic APPRIS ALT1 Cpsf6- ENSMUST00000175888.1 449 39aa ENSMUSP00000135840.1 Protein coding - H3BLM1 CDS 5' 202 incomplete TSL:3 Cpsf6- ENSMUST00000175924.1 377 91aa ENSMUSP00000135848.1 Protein coding - H3BLM9 CDS 3' 203 incomplete TSL:5 Cpsf6- ENSMUST00000176480.7 717 182aa ENSMUSP00000135550.1 Nonsense mediated - H3BKW0 CDS 5' 204 decay incomplete TSL:3 Cpsf6- ENSMUST00000176567.1 2749 No - Retained intron - - TSL:2 205 protein Page 7 of 9 https://www.alphaknockout.com 55.34 kb Forward strand 117.34Mb 117.35Mb 117.36Mb 117.37Mb 117.38Mb 117.39Mb Contigs < AC123694.11 Genes (Comprehensive set... < Cpsf6-201protein coding < Cpsf6-207protein coding < Cpsf6-206protein coding < Cpsf6-204nonsense mediated decay < Cpsf6-203protein coding < Cpsf6-208protein coding < Cpsf6-202protein coding < Cpsf6-205retained intron Regulatory Build 117.34Mb 117.35Mb 117.36Mb 117.37Mb 117.38Mb 117.39Mb Reverse strand 55.34 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding
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