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Mouse Qpct Knockout Project (CRISPR/Cas9)

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Mouse Qpct Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Qpct Knockout Project (CRISPR/Cas9) Objective: To create a Qpct knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Qpct gene (NCBI Reference Sequence: NM_027455 ; Ensembl: ENSMUSG00000024084 ) is located on Mouse chromosome 17. 7 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 7 (Transcript: ENSMUST00000040789). Exon 3~5 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 exhibit normal motor coordination and spatial working memory. Exon 3 starts from about 24.95% of the coding region. Exon 3~5 covers 51.2% of the coding region. The size of effective KO region: ~8894 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 3 4 5 7 Legends Exon of mouse Qpct 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 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 2000 bp section downstream of Exon 5 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(27.25% 545) | C(21.15% 423) | T(28.2% 564) | G(23.4% 468) Note: The 2000 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(2000bp) | A(22.4% 448) | C(24.95% 499) | T(33.3% 666) | G(19.35% 387) Note: The 2000 bp section downstream of Exon 5 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% chr17 + 79068674 79070673 2000 browser details YourSeq 33 1960 1993 2000 100.0% chr3 + 63116451 63116486 36 browser details YourSeq 32 530 574 2000 91.9% chr1 - 88767513 88767568 56 browser details YourSeq 30 1965 1994 2000 100.0% chr1 - 139981489 139981518 30 browser details YourSeq 29 1963 1993 2000 90.0% chr1 - 105861461 105861490 30 browser details YourSeq 29 1810 1848 2000 87.2% chr3 + 143748003 143748041 39 browser details YourSeq 24 1976 2000 2000 100.0% chr1 - 39751942 39752438 497 browser details YourSeq 22 1799 1820 2000 100.0% chr13 + 34316085 34316106 22 browser details YourSeq 21 1674 1694 2000 100.0% chr2 - 5323801 5323821 21 browser details YourSeq 21 1972 1992 2000 100.0% chr1 - 38591611 38591631 21 browser details YourSeq 21 901 921 2000 100.0% chr15 + 102572391 102572411 21 Note: The 2000 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 2000 1 2000 2000 100.0% chr17 + 79079568 79081567 2000 browser details YourSeq 413 1149 1804 2000 86.2% chr1 - 58707437 58708073 637 browser details YourSeq 399 1199 1804 2000 86.3% chr6 - 53936577 53937200 624 browser details YourSeq 389 1186 1766 2000 85.6% chr6 - 135330494 135331083 590 browser details YourSeq 384 1207 1804 2000 85.9% chr1 - 195063887 195064495 609 browser details YourSeq 382 1199 1804 2000 87.2% chr8 + 40686560 40687167 608 browser details YourSeq 380 1204 1803 2000 86.9% chr2 - 114893050 114893679 630 browser details YourSeq 378 1199 1770 2000 87.9% chr6 + 141672720 141673531 812 browser details YourSeq 377 1189 1777 2000 86.7% chr8 + 82704247 82704831 585 browser details YourSeq 374 1203 1801 2000 85.9% chr8 + 107309028 107309625 598 browser details YourSeq 372 1187 1800 2000 84.7% chr12 - 101130649 101131279 631 browser details YourSeq 372 1189 1804 2000 88.6% chr9 + 90401974 90402596 623 browser details YourSeq 372 1063 1804 2000 85.5% chr18 + 31452255 31452896 642 browser details YourSeq 369 1200 1777 2000 85.6% chr8 + 12880907 12881495 589 browser details YourSeq 362 1191 1775 2000 87.7% chr19 + 41037986 41038573 588 browser details YourSeq 361 1199 1741 2000 88.1% chr16 - 87925390 87925934 545 browser details YourSeq 360 1199 1802 2000 86.2% chr2 - 78382487 78383093 607 browser details YourSeq 358 1187 1804 2000 86.6% chrX - 13778594 13779230 637 browser details YourSeq 358 1189 1804 2000 85.6% chrX + 57771942 57772591 650 browser details YourSeq 358 1180 1766 2000 86.4% chr12 + 61137292 61137893 602 Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Qpct glutaminyl-peptide cyclotransferase (glutaminyl cyclase) [ Mus musculus (house mouse) ] Gene ID: 70536, updated on 24-Oct-2019 Gene summary Official Symbol Qpct provided by MGI Official Full Name glutaminyl-peptide cyclotransferase (glutaminyl cyclase) provided by MGI Primary source MGI:MGI:1917786 See related Ensembl:ENSMUSG00000024084 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 5730422A13Rik Expression Broad expression in liver E18 (RPKM 17.4), testis adult (RPKM 10.4) and 23 other tissues See more Orthologs human all Genomic context Location: 17; 17 E3 See Qpct in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (79051906..79090378) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (79451246..79489583) Chromosome 17 - NC_000083.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Qpct ENSMUSG00000024084 Description glutaminyl-peptide cyclotransferase (glutaminyl cyclase) [Source:MGI Symbol;Acc:MGI:1917786] Gene Synonyms 5730422A13Rik Location Chromosome 17: 79,051,906-79,090,378 forward strand. GRCm38:CM001010.2 About this gene This gene has 3 transcripts (splice variants), 201 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Qpct-201 ENSMUST00000040789.5 2059 362aa ENSMUSP00000038732.4 Protein coding CCDS28983 A0A0R4J0G6 TSL:1 GENCODE basic APPRIS P2 Qpct-203 ENSMUST00000233916.1 1473 313aa ENSMUSP00000156493.1 Protein coding - A0A3B2W7G1 GENCODE basic APPRIS ALT2 Qpct-202 ENSMUST00000233899.1 864 No protein - Retained intron - - - 58.47 kb Forward strand 79.05Mb 79.06Mb 79.07Mb 79.08Mb 79.09Mb 79.10Mb Genes (Comprehensive set... Gm26957-201 >processed pseudogene Qpct-202 >retained intron Qpct-201 >protein coding Qpct-203 >protein coding Contigs < AC151293.2 Regulatory Build 79.05Mb 79.06Mb 79.07Mb 79.08Mb 79.09Mb 79.10Mb Reverse strand 58.47 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 Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000040789 38.47 kb Forward strand Qpct-201 >protein coding ENSMUSP00000038... Low complexity (Seg) Superfamily SSF53187 Pfam Peptidase M28 PANTHER PTHR12283 PTHR12283:SF5 Gene3D 3.40.630.10 CDD M28 Zn-Peptidase Glutaminyl Cyclase All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 362 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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