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Mouse Vps45 Knockout Project (CRISPR/Cas9)

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Mouse Vps45 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Vps45 Knockout Project (CRISPR/Cas9) Objective: To create a Vps45 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Vps45 gene (NCBI Reference Sequence: NM_013841 ; Ensembl: ENSMUSG00000015747 ) is located on Mouse chromosome 3. 15 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 15 (Transcript: ENSMUST00000015891). Exon 2~3 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 5.5% of the coding region. Exon 2~3 covers 11.46% of the coding region. The size of effective KO region: ~4076 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 15 Legends Exon of mouse Vps45 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 567 bp section upstream of Exon 2 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 2000 bp section downstream 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(567bp) | A(27.87% 158) | C(16.75% 95) | T(34.04% 193) | G(21.34% 121) Note: The 567 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(29.4% 588) | C(21.35% 427) | T(29.8% 596) | G(19.45% 389) Note: The 2000 bp section downstream 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. 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 567 1 567 567 100.0% chr3 - 96057115 96057681 567 browser details YourSeq 33 33 98 567 97.2% chr1 + 97615741 97685304 69564 browser details YourSeq 21 19 39 567 100.0% chr10 + 88090649 88090669 21 browser details YourSeq 21 400 421 567 100.0% chr10 + 10179281 10179303 23 browser details YourSeq 20 453 474 567 95.5% chr13 - 93176601 93176622 22 browser details YourSeq 20 441 460 567 100.0% chr14 + 85502571 85502590 20 Note: The 567 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% chr3 - 96051039 96053038 2000 browser details YourSeq 185 146 610 2000 85.5% chr5 + 147818191 147818757 567 browser details YourSeq 156 190 610 2000 89.4% chr4 + 140951167 140951632 466 browser details YourSeq 153 203 610 2000 90.0% chr1 + 135275115 135275719 605 browser details YourSeq 148 411 616 2000 86.3% chr4 - 57919170 57919365 196 browser details YourSeq 148 438 1080 2000 82.0% chr19 - 23115749 23116215 467 browser details YourSeq 146 428 615 2000 90.3% chrX - 142384173 142384359 187 browser details YourSeq 146 412 610 2000 91.0% chr2 + 38441311 38441513 203 browser details YourSeq 144 190 614 2000 87.5% chr12 - 86827845 86828323 479 browser details YourSeq 141 428 615 2000 89.4% chr6 - 52484020 52484205 186 browser details YourSeq 140 188 610 2000 90.7% chr18 + 32134064 32134487 424 browser details YourSeq 139 428 769 2000 82.7% chr17 - 85054153 85054404 252 browser details YourSeq 138 270 610 2000 82.4% chr9 - 102707660 102707881 222 browser details YourSeq 137 428 614 2000 88.5% chr5 - 99776422 99776607 186 browser details YourSeq 137 206 610 2000 86.4% chr17 - 37872936 37873435 500 browser details YourSeq 136 431 610 2000 91.0% chr5 + 143912086 143912267 182 browser details YourSeq 136 428 615 2000 86.6% chr10 + 90566977 90567159 183 browser details YourSeq 134 428 614 2000 88.1% chr9 - 22214333 22214518 186 browser details YourSeq 134 428 615 2000 87.4% chr6 - 52598825 52599011 187 browser details YourSeq 134 428 615 2000 85.8% chr4 - 151027428 151027608 181 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Vps45 vacuolar protein sorting 45 [ Mus musculus (house mouse) ] Gene ID: 22365, updated on 12-Aug-2019 Gene summary Official Symbol Vps45 provided by MGI Official Full Name vacuolar protein sorting 45 provided by MGI Primary source MGI:MGI:891965 See related Ensembl:ENSMUSG00000015747 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 mVps45; AI462172; AW554165 Expression Ubiquitous expression in CNS E18 (RPKM 3.3), CNS E14 (RPKM 3.0) and 28 other tissues See more Orthologs human all Genomic context Location: 3 F2.1; 3 41.65 cM See Vps45 in Genome Data Viewer Exon count: 16 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (95999832..96059661, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (95803755..95862378, complement) Chromosome 3 - NC_000069.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Vps45 ENSMUSG00000015747 Description vacuolar protein sorting 45 [Source:MGI Symbol;Acc:MGI:891965] Gene Synonyms mVps45 Location Chromosome 3: 95,999,832-96,058,466 reverse strand. GRCm38:CM000996.2 About this gene This gene has 3 transcripts (splice variants), 198 orthologues, 7 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Vps45-201 ENSMUST00000015891.5 2637 570aa ENSMUSP00000015891.5 Protein coding CCDS17628 P97390 TSL:1 GENCODE basic APPRIS P1 Vps45-202 ENSMUST00000131281.1 2004 No protein - Retained intron - - TSL:1 Vps45-203 ENSMUST00000140518.1 266 No protein - lncRNA - - TSL:3 78.64 kb Forward strand 96.00Mb 96.02Mb 96.04Mb 96.06Mb Contigs AC092855.39 > Genes (Comprehensive set... < Plekho1-201protein coding < Vps45-202retained intron < Plekho1-202protein coding < Vps45-203lncRNA < Plekho1-203protein coding < Plekho1-204protein coding < Plekho1-205retained intron < Vps45-201protein coding Regulatory Build 96.00Mb 96.02Mb 96.04Mb 96.06Mb Reverse strand 78.64 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 processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000015891 < Vps45-201protein coding Reverse strand 58.63 kb ENSMUSP00000015... Low complexity (Seg) Superfamily Sec1-like superfamily Pfam Sec1-like protein PIRSF Sec1-like protein PANTHER PTHR11679:SF3 Sec1-like protein Gene3D 3.40.50.2060 3.90.830.10 1.25.40.60 Sec1-like, domain 2 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 570 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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