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Mouse Ddx24 Knockout Project (CRISPR/Cas9)

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Mouse Ddx24 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ddx24 Knockout Project (CRISPR/Cas9) Objective: To create a Ddx24 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ddx24 gene (NCBI Reference Sequence: NM_001159502 ; Ensembl: ENSMUSG00000041645 ) is located on Mouse chromosome 12. 9 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000110001). Exon 2~6 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: Homozygous knockout is embryonic lethal: embryos die between implantation and placentation. Heterozygous KO animals are viable and fertile. Exon 2 starts from about 4.95% of the coding region. Exon 2~6 covers 73.61% of the coding region. The size of effective KO region: ~8402 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 9 Legends Exon of mouse Ddx24 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 928 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 6 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(928bp) | A(23.71% 220) | C(22.63% 210) | T(29.53% 274) | G(24.14% 224) Note: The 928 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(27.05% 541) | C(23.0% 460) | T(29.45% 589) | G(20.5% 410) Note: The 2000 bp section downstream of Exon 6 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 928 1 928 928 100.0% chr12 - 103424598 103425525 928 browser details YourSeq 47 295 355 928 94.5% chr15 - 51525955 51526039 85 browser details YourSeq 43 301 349 928 96.0% chr19 + 3368252 3368327 76 browser details YourSeq 36 316 359 928 93.2% chr2 - 35485701 35485746 46 browser details YourSeq 34 312 345 928 100.0% chr11 - 76447076 76447109 34 browser details YourSeq 34 289 342 928 73.7% chr14 + 20418560 20418601 42 browser details YourSeq 33 341 499 928 97.3% chr6 + 71145067 71145227 161 browser details YourSeq 33 312 348 928 94.6% chr1 + 169484641 169484677 37 browser details YourSeq 32 314 354 928 90.0% chr8 + 72670671 72670718 48 browser details YourSeq 31 314 348 928 94.3% chr11 - 68328187 68328221 35 browser details YourSeq 30 322 354 928 97.0% chr2 - 164789837 164789876 40 browser details YourSeq 30 311 348 928 89.5% chr4 + 136731006 136731043 38 browser details YourSeq 29 296 327 928 96.8% chr11 - 88267801 88267834 34 browser details YourSeq 29 316 348 928 94.0% chr2 + 103993380 103993412 33 browser details YourSeq 28 313 342 928 96.7% chr7 - 78828715 78828744 30 browser details YourSeq 28 311 344 928 91.2% chr17 - 73090296 73090329 34 browser details YourSeq 28 303 334 928 93.8% chr14 + 54452511 54452542 32 browser details YourSeq 27 296 334 928 84.7% chr6 - 97038381 97038419 39 browser details YourSeq 27 316 344 928 96.6% chr14 - 55010894 55010922 29 browser details YourSeq 27 314 344 928 93.6% chr1 - 131866000 131866030 31 Note: The 928 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% chr12 - 103414196 103416195 2000 browser details YourSeq 182 779 1502 2000 82.0% chr12 - 116209839 116210620 782 browser details YourSeq 179 874 1598 2000 88.6% chr6 + 86953554 86954397 844 browser details YourSeq 177 902 1389 2000 89.8% chr4 - 103244772 103245352 581 browser details YourSeq 177 810 1392 2000 84.3% chrX + 98973123 98973889 767 browser details YourSeq 161 984 1592 2000 80.7% chr4 + 40565936 40566514 579 browser details YourSeq 154 954 1517 2000 88.6% chr13 - 23824490 23825098 609 browser details YourSeq 141 1210 1689 2000 81.3% chr15 - 95166059 95166548 490 browser details YourSeq 139 1156 1703 2000 79.6% chr1 + 82217804 82218217 414 browser details YourSeq 138 981 1707 2000 85.6% chrX - 69217533 69218304 772 browser details YourSeq 136 631 1124 2000 80.7% chr12 - 72314660 72315131 472 browser details YourSeq 131 1188 1611 2000 87.1% chr7 + 133302526 133302955 430 browser details YourSeq 122 910 1254 2000 91.4% chr4 - 58721651 58722041 391 browser details YourSeq 122 673 1049 2000 80.3% chr6 + 18556446 18556767 322 browser details YourSeq 120 1015 1700 2000 82.0% chr2 + 91872177 91872829 653 browser details YourSeq 118 1225 1686 2000 80.7% chr1 + 47033895 47034536 642 browser details YourSeq 117 769 1073 2000 80.8% chr1 - 162747371 162747655 285 browser details YourSeq 117 790 1259 2000 80.0% chr1 + 94214013 94214497 485 browser details YourSeq 115 1357 1826 2000 89.1% chr18 - 57396107 57396578 472 browser details YourSeq 111 880 1088 2000 89.3% chr18 - 46070089 46070297 209 Note: The 2000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Ddx24 DEAD (Asp-Glu-Ala-Asp) box polypeptide 24 [ Mus musculus (house mouse) ] Gene ID: 27225, updated on 24-Oct-2019 Gene summary Official Symbol Ddx24 provided by MGI Official Full Name DEAD (Asp-Glu-Ala-Asp) box polypeptide 24 provided by MGI Primary source MGI:MGI:1351337 See related Ensembl:ENSMUSG00000041645 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 AI649272; 1700055J08Rik; 2510027P10Rik Expression Ubiquitous expression in cerebellum adult (RPKM 38.5), cortex adult (RPKM 31.2) and 28 other tissues See more Orthologs human all Genomic context Location: 12; 12 E See Ddx24 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (103407976..103425847, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (104646186..104664077, complement) Chromosome 12 - NC_000078.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Ddx24 ENSMUSG00000041645 Description DEAD (Asp-Glu-Ala-Asp) box polypeptide 24 [Source:MGI Symbol;Acc:MGI:1351337] Gene Synonyms 1700055J08Rik, 2510027P10Rik Location Chromosome 12: 103,407,982-103,425,830 reverse strand. GRCm38:CM001005.2 About this gene This gene has 9 transcripts (splice variants), 207 orthologues, 39 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ddx24- ENSMUST00000110001.3 3063 903aa ENSMUSP00000105628.2 Protein coding CCDS49152 F8WJA0 TSL:1 202 GENCODE basic APPRIS ALT2 Ddx24- ENSMUST00000044923.14 2850 857aa ENSMUSP00000040890.7 Protein coding CCDS26129 Q9ESV0 TSL:1 201 GENCODE basic APPRIS P3 Ddx24- ENSMUST00000221211.1 2669 791aa ENSMUSP00000152206.1 Protein coding - A0A1Y7VKX7 TSL:1 206 GENCODE basic APPRIS ALT2 Ddx24- ENSMUST00000222782.1 527 176aa ENSMUSP00000152745.1 Protein coding - A0A1Y7VK75 CDS 5' and 3' 208 incomplete TSL:5 Ddx24- ENSMUST00000223233.1 485 119aa ENSMUSP00000152058.1 Protein coding - A0A1Y7VMS6 CDS 3' incomplete 209 TSL:3 Ddx24- ENSMUST00000220975.1 561 119aa ENSMUSP00000152366.1 Non stop - A0A1Y7VLU4 TSL:1 205 decay Ddx24- ENSMUST00000220705.1 4274 No - Retained - - TSL:1 204 protein intron Ddx24- ENSMUST00000222715.1 1646 No - Retained - - TSL:NA 207 protein intron Ddx24- ENSMUST00000220481.1 386 No - Retained - - TSL:3 203 protein intron Page 7 of 9 https://www.alphaknockout.com 37.85 kb Forward strand 103.40Mb 103.41Mb 103.42Mb 103.43Mb Genes Otub2-202 >protein coding Ifi27-206 >retained intron (Comprehensive set... Otub2-201 >protein coding Ifi27-208 >retained intron Otub2-203 >retained intron Ifi27-202 >protein coding Otub2-204 >protein coding Ifi27-203 >protein coding Otub2-205 >protein coding Ifi27-204 >protein coding Ifi27-201 >protein coding Ifi27-205 >protein coding Ifi27-207 >protein coding Contigs AC154347.2 > Genes (Comprehensive set... < 9330161L09Rik-201TEC < Ddx24-204retained intron < 9330161L09Rik-202TEC < Ddx24-205non stop decay < Ddx24-201protein coding < Ddx24-202protein coding < Ddx24-208protein coding < Ddx24-209protein coding < Ddx24-207retained intron < Ddx24-206protein coding < Ddx24-203retained intron < Gm47260-201TEC Regulatory Build 103.40Mb 103.41Mb 103.42Mb 103.43Mb Reverse strand 37.85 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000110001 < Ddx24-202protein coding Reverse strand 17.80 kb ENSMUSP00000105..
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