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Mouse Dhx32 Knockout Project (CRISPR/Cas9)

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Mouse Dhx32 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Dhx32 Knockout Project (CRISPR/Cas9) Objective: To create a Dhx32 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Dhx32 gene (NCBI Reference Sequence: NM_133941 ; Ensembl: ENSMUSG00000030986 ) is located on Mouse chromosome 7. 12 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000033290). Exon 4~7 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 4 starts from about 22.1% of the coding region. Exon 4~7 covers 38.97% of the coding region. The size of effective KO region: ~8834 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 4 5 6 7 12 Legends Exon of mouse Dhx32 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 4 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 7 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(28.1% 562) | C(20.95% 419) | T(27.4% 548) | G(23.55% 471) Note: The 2000 bp section upstream of Exon 4 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(22.6% 452) | C(23.2% 464) | T(28.3% 566) | G(25.9% 518) Note: The 2000 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 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% chr7 - 133742802 133744801 2000 browser details YourSeq 59 455 574 2000 90.5% chr3 + 131478388 131478528 141 browser details YourSeq 56 515 592 2000 82.9% chr11 - 86228625 86228698 74 browser details YourSeq 53 501 584 2000 93.5% chr7 - 80752410 80822082 69673 browser details YourSeq 52 529 589 2000 93.5% chr11 - 98006769 98006830 62 browser details YourSeq 52 472 575 2000 85.2% chr2 + 161104170 161104296 127 browser details YourSeq 51 501 573 2000 87.0% chr19 + 53691865 53691945 81 browser details YourSeq 50 520 592 2000 88.1% chr9 - 67576247 67576319 73 browser details YourSeq 50 504 573 2000 85.8% chr11 + 85878499 85878568 70 browser details YourSeq 49 456 572 2000 86.6% chr17 - 24670853 24671089 237 browser details YourSeq 49 464 575 2000 85.6% chr14 + 50769789 50770293 505 browser details YourSeq 45 530 583 2000 92.6% chr7 + 81486919 81486973 55 browser details YourSeq 45 514 579 2000 84.9% chr10 + 91055586 91055652 67 browser details YourSeq 43 514 566 2000 90.6% chr14 - 50848628 50848680 53 browser details YourSeq 43 513 569 2000 87.8% chr11 - 73259085 73259141 57 browser details YourSeq 43 499 573 2000 78.7% chr1 + 153433682 153433756 75 browser details YourSeq 42 524 575 2000 90.4% chr11 - 46989015 46989066 52 browser details YourSeq 42 514 573 2000 85.0% chr1 - 177290647 177290706 60 browser details YourSeq 42 520 576 2000 87.8% chr18 + 65513974 65514031 58 browser details YourSeq 42 502 575 2000 81.7% chr12 + 3394856 3394925 70 Note: The 2000 bp section upstream of Exon 4 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% chr7 - 133731968 133733967 2000 browser details YourSeq 226 318 702 2000 87.5% chr9 + 40727686 40932550 204865 browser details YourSeq 226 316 705 2000 84.3% chr5 + 149093910 149094299 390 browser details YourSeq 212 335 715 2000 85.7% chr1 + 36752936 36753297 362 browser details YourSeq 205 331 947 2000 82.4% chr8 + 11306186 11306690 505 browser details YourSeq 203 322 705 2000 86.7% chr17 - 68007997 68008382 386 browser details YourSeq 202 324 994 2000 82.4% chr2 + 33675148 33675656 509 browser details YourSeq 201 316 705 2000 87.9% chr11 + 50838727 50839123 397 browser details YourSeq 200 332 711 2000 86.1% chr12 - 17560316 17560684 369 browser details YourSeq 194 342 705 2000 79.5% chr3 - 102037720 102038082 363 browser details YourSeq 193 317 704 2000 84.5% chr18 - 56364306 56364683 378 browser details YourSeq 192 318 683 2000 87.1% chr7 + 129637551 129637919 369 browser details YourSeq 192 322 694 2000 86.1% chr15 + 31666758 31667133 376 browser details YourSeq 192 322 705 2000 84.0% chr12 + 75863115 75863499 385 browser details YourSeq 190 331 693 2000 88.8% chr5 + 37182465 37182828 364 browser details YourSeq 190 342 704 2000 89.4% chr10 + 119689996 119815299 125304 browser details YourSeq 188 318 679 2000 87.5% chr18 + 74968906 74969273 368 browser details YourSeq 187 370 687 2000 84.4% chr7 - 37520957 37521273 317 browser details YourSeq 186 219 697 2000 80.2% chr18 - 66173893 66174593 701 browser details YourSeq 184 404 704 2000 80.4% chr10 - 116624598 116624894 297 Note: The 2000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Dhx32 DEAH (Asp-Glu-Ala-His) box polypeptide 32 [ Mus musculus (house mouse) ] Gene ID: 101437, updated on 10-Oct-2019 Gene summary Official Symbol Dhx32 provided by MGI Official Full Name DEAH (Asp-Glu-Ala-His) box polypeptide 32 provided by MGI Primary source MGI:MGI:2141813 See related Ensembl:ENSMUSG00000030986 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 Ddx32; muDDX32; AA408140; 3110079L04Rik; 4732469F02Rik Expression Ubiquitous expression in subcutaneous fat pad adult (RPKM 23.6), placenta adult (RPKM 18.1) and 28 other tissues See Orthologs more human all Genomic context Location: 7; 7 F3 See Dhx32 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (133720935..133782785, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (140912618..140968486, complement) Chromosome 7 - NC_000073.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 10 transcripts Gene: Dhx32 ENSMUSG00000030986 Description DEAH (Asp-Glu-Ala-His) box polypeptide 32 [Source:MGI Symbol;Acc:MGI:2141813] Gene Synonyms Ddx32 Location Chromosome 7: 133,720,942-133,782,726 reverse strand. GRCm38:CM001000.2 About this gene This gene has 10 transcripts (splice variants), 242 orthologues, 18 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Dhx32-201 ENSMUST00000033290.11 2955 751aa ENSMUSP00000033290.5 Protein coding CCDS21936 Q8BZS9 TSL:1 GENCODE basic APPRIS P1 Dhx32-202 ENSMUST00000063669.7 2644 751aa ENSMUSP00000066067.1 Protein coding CCDS21936 Q8BZS9 TSL:1 GENCODE basic APPRIS P1 Dhx32-203 ENSMUST00000106139.7 2233 611aa ENSMUSP00000101745.1 Protein coding CCDS72060 D3Z4E3 TSL:1 GENCODE basic Dhx32-204 ENSMUST00000130182.1 769 110aa ENSMUSP00000115677.1 Protein coding - D3YZD7 CDS 3' incomplete TSL:3 Dhx32-205 ENSMUST00000135989.1 604 8aa ENSMUSP00000118326.1 Protein coding - A0A1C7ZMZ3 CDS 3' incomplete TSL:3 Dhx32-209 ENSMUST00000149876.1 558 143aa ENSMUSP00000121789.1 Protein coding - D3Z255 CDS 3' incomplete TSL:2 Dhx32-208 ENSMUST00000146211.1 546 No protein - Retained intron - - TSL:1 Dhx32-207 ENSMUST00000140593.8 2998 No protein - lncRNA - - TSL:5 Dhx32-206 ENSMUST00000136301.1 698 No protein - lncRNA - - TSL:2 Dhx32-210 ENSMUST00000211450.1 673 No protein - lncRNA - - TSL:5 Page 7 of 9 https://www.alphaknockout.com 81.78 kb Forward strand Genes Bccip-201 >protein coding Gm45670-201 >lncRNA Fank1-201 >protein coding (Comprehensive set... Bccip-205 >retained intron Fank1-204 >protein coding Bccip-202 >retained intron Fank1-203 >protein coding Bccip-204 >lncRNA Fank1-202 >protein coding Bccip-203 >retained intron Fank1-205 >protein coding Contigs < AC127348.4 Genes (Comprehensive set... < Dhx32-201protein coding < Dhx32-207lncRNA < Dhx32-203protein coding < Dhx32-202protein coding < Dhx32-206lncRNA < Gm15483-201processed pseudogene < Dhx32-205protein coding < Dhx32-210lncRNA < Dhx32-208retained intron < Dhx32-209protein coding < Dhx32-204protein coding Regulatory Build Reverse strand 81.78 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene pseudogene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000033290 < Dhx32-201protein coding Reverse strand 55.86 kb ENSMUSP00000033... MobiDB lite Low complexity (Seg) Superfamily P-loop containing nucleoside triphosphate hydrolase SMART Helicase-associated domain Pfam Helicase-associated domain Domain of unknown function DUF1605 PANTHER PTHR18934 PTHR18934:SF88 Gene3D 3.40.50.300 1.20.120.1080 CDD cd17977 cd18791 All sequence SNPs/i..
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