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Mouse Pcsk7 Knockout Project (CRISPR/Cas9)

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Mouse Pcsk7 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pcsk7 Knockout Project (CRISPR/Cas9) Objective: To create a Pcsk7 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pcsk7 gene (NCBI Reference Sequence: NM_008794 ; Ensembl: ENSMUSG00000035382 ) is located on Mouse chromosome 9. 17 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 17 (Transcript: ENSMUST00000039059). Exon 3~8 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: Mice homozygous for a knock-out allele exhibit normal response to LPS. Exon 3 starts from the coding region. Exon 3~8 covers 45.5% of the coding region. The size of effective KO region: ~5967 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 3 4 5 6 7 8 17 Legends Exon of mouse Pcsk7 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 608 bp section upstream of Exon 3 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 761 bp section downstream of Exon 8 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 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(608bp) | A(26.32% 160) | C(17.76% 108) | T(25.66% 156) | G(30.26% 184) Note: The 608 bp section upstream of Exon 3 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(761bp) | A(22.34% 170) | C(24.97% 190) | T(33.25% 253) | G(19.45% 148) Note: The 761 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 608 1 608 608 100.0% chr9 + 45908665 45909272 608 browser details YourSeq 77 129 238 608 93.2% chr8 - 61793916 61794025 110 browser details YourSeq 75 109 233 608 89.4% chr15 - 34354048 34354197 150 browser details YourSeq 73 115 238 608 93.0% chr10 + 93158126 93158322 197 browser details YourSeq 72 91 206 608 80.5% chr17 + 24562676 24562783 108 browser details YourSeq 69 67 175 608 91.1% chr17 + 35132759 35132866 108 browser details YourSeq 66 66 182 608 90.2% chr6 + 59412136 59412253 118 browser details YourSeq 66 107 190 608 94.7% chr16 + 10979056 11033795 54740 browser details YourSeq 63 64 173 608 91.9% chr7 - 16025456 16125266 99811 browser details YourSeq 61 66 173 608 90.7% chr11 + 70975178 70990842 15665 browser details YourSeq 59 109 186 608 80.0% chr5 - 123198137 123198206 70 browser details YourSeq 58 129 206 608 81.9% chr3 + 145786906 145786975 70 browser details YourSeq 57 113 190 608 90.3% chr14 - 31281579 31281658 80 browser details YourSeq 57 109 189 608 83.7% chr5 + 3572138 3572209 72 browser details YourSeq 56 109 189 608 85.8% chr10 - 43041296 43041374 79 browser details YourSeq 56 124 190 608 85.0% chr5 + 77028730 77028789 60 browser details YourSeq 55 109 173 608 87.1% chrX - 60947286 60947347 62 browser details YourSeq 55 120 190 608 86.3% chr12 - 3662217 3662280 64 browser details YourSeq 55 125 217 608 91.6% chr1 - 192233811 192233901 91 browser details YourSeq 54 113 190 608 79.7% chr12 + 87354071 87354139 69 Note: The 608 bp section upstream of Exon 3 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 761 1 761 761 100.0% chr9 + 45915228 45915988 761 browser details YourSeq 26 551 577 761 100.0% chr10 - 77175441 77175868 428 browser details YourSeq 21 685 705 761 100.0% chr14 + 57930188 57930208 21 browser details YourSeq 20 326 347 761 95.5% chr12 - 96802516 96802537 22 browser details YourSeq 20 322 341 761 100.0% chr10 - 11674509 11674528 20 browser details YourSeq 20 637 656 761 100.0% chr1 + 43663915 43663934 20 Note: The 761 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: Pcsk7 proprotein convertase subtilisin/kexin type 7 [ Mus musculus (house mouse) ] Gene ID: 18554, updated on 10-Oct-2019 Gene summary Official Symbol Pcsk7 provided by MGI Official Full Name proprotein convertase subtilisin/kexin type 7 provided by MGI Primary source MGI:MGI:107421 See related Ensembl:ENSMUSG00000035382 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 PC7; SPC7; AA959856 Expression Biased expression in bladder adult (RPKM 616.5), stomach adult (RPKM 205.4) and 7 other tissues See more Orthologs human all Genomic context Location: 9 A5.2; 9 25.32 cM See Pcsk7 in Genome Data Viewer Exon count: 19 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (45906447..45932107) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (45714652..45737806) Chromosome 9 - NC_000075.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Pcsk7 ENSMUSG00000035382 Description proprotein convertase subtilisin/kexin type 7 [Source:MGI Symbol;Acc:MGI:107421] Gene Synonyms SPC7 Location Chromosome 9: 45,906,497-45,929,726 forward strand. GRCm38:CM001002.2 About this gene This gene has 9 transcripts (splice variants), 197 orthologues, 9 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pcsk7- ENSMUST00000039059.7 3876 770aa ENSMUSP00000047508.6 Protein coding CCDS40609 Q61139 TSL:1 201 GENCODE basic APPRIS P1 Pcsk7- ENSMUST00000216672.1 3599 770aa ENSMUSP00000150393.1 Protein coding CCDS40609 Q61139 TSL:1 209 GENCODE basic APPRIS P1 Pcsk7- ENSMUST00000215189.1 3893 518aa ENSMUSP00000150500.1 Nonsense mediated - A0A1L1STW0 TSL:1 204 decay Pcsk7- ENSMUST00000213854.1 547 86aa ENSMUSP00000150379.1 Nonsense mediated - A0A1L1STL8 CDS 5' 202 decay incomplete TSL:3 Pcsk7- ENSMUST00000215535.1 935 No - Retained intron - - TSL:NA 205 protein Pcsk7- ENSMUST00000214425.1 749 No - Retained intron - - TSL:2 203 protein Pcsk7- ENSMUST00000216514.1 608 No - Retained intron - - TSL:3 207 protein Pcsk7- ENSMUST00000216614.1 442 No - lncRNA - - TSL:5 208 protein Pcsk7- ENSMUST00000216504.1 229 No - lncRNA - - TSL:1 206 protein Page 7 of 9 https://www.alphaknockout.com 43.23 kb Forward strand 45.90Mb 45.91Mb 45.92Mb 45.93Mb Genes (Comprehensive set... Pcsk7-201 >protein coding Gm48529-201 >lncRNA Pcsk7-205 >retained intron Pcsk7-208 >lncRNA Pcsk7-203 >retained intron Pcsk7-204 >nonsense mediated decay Gm48529-202 >lncRNA Pcsk7-209 >protein coding Pcsk7-202 >nonsense mediated decay Pcsk7-206 >lncRNA Pcsk7-207 >retained intron Contigs AC126804.3 > < AC122273.4 Genes < Rnf214-209protein coding < Tagln-201protein coding (Comprehensive set... < Rnf214-201protein coding < Tagln-203retained intron < Rnf214-208protein coding < Tagln-202protein coding < Rnf214-210nonsense mediated decay < Sidt2-201protein coding < Rnf214-211nonsense mediated decay < Sidt2-202protein coding < Rnf214-205protein coding < Sidt2-203protein coding < Rnf214-212nonsense mediated decay < Mir7087-201miRNA < Rnf214-204retained intron < Rnf214-206nonsense mediated decay < Rnf214-203retained intron < Rnf214-213lncRNA Regulatory Build 45.90Mb 45.91Mb 45.92Mb 45.93Mb Reverse strand 43.23 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000039059 23.22 kb Forward strand Pcsk7-201 >protein coding ENSMUSP00000047... Transmembrane heli... Low complexity (Seg) Superfamily SSF54897 Peptidase S8/S53 domain superfamily Galactose-binding-like domain superfamily Prints Peptidase S8, subtilisin-related Pfam Peptidase S8/S53 domain P domain Peptidase S8, pro-domain PROSITE profiles P domain PROSITE patterns Peptidase S8, subtilisin, His-active site Peptidase S8, subtilisin, Ser-active site PANTHER PTHR42884 PTHR42884:SF17 Gene3D Peptidase S8, pro-domain superfamily Galactose-binding-like domain superfamily Peptidase S8/S53 domain superfamily CDD Kexin/furin catalytic domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 770 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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