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Mouse Dctn4 Knockout Project (CRISPR/Cas9)

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Mouse Dctn4 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Dctn4 Knockout Project (CRISPR/Cas9) Objective: To create a Dctn4 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Dctn4 gene (NCBI Reference Sequence: NM_026302 ; Ensembl: ENSMUSG00000024603 ) is located on Mouse chromosome 18. 13 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 13 (Transcript: ENSMUST00000025505). 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 9.86% of the coding region. Exon 2~3 covers 18.12% of the coding region. The size of effective KO region: ~3850 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 13 Legends Exon of mouse Dctn4 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 2 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. 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. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. 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(27.15% 543) | C(24.45% 489) | T(28.0% 560) | G(20.4% 408) Note: The 2000 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(25.5% 510) | C(16.6% 332) | T(34.8% 696) | G(23.1% 462) 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 2000 1 2000 2000 100.0% chr18 + 60532594 60534593 2000 browser details YourSeq 1084 1 1129 2000 98.4% chr7 + 128608874 128610061 1188 browser details YourSeq 1074 1 1129 2000 98.3% chr2 - 111860994 111862200 1207 browser details YourSeq 1043 1 1130 2000 98.0% chr1 - 139724768 139725899 1132 browser details YourSeq 1042 1 1130 2000 97.9% chr2 + 17751589 17752755 1167 browser details YourSeq 1028 1 1130 2000 98.3% chr15 + 37096277 37097424 1148 browser details YourSeq 1023 63 1130 2000 98.5% chr13 - 103929944 103931150 1207 browser details YourSeq 1021 63 1129 2000 98.7% chr7 - 137894370 137895578 1209 browser details YourSeq 1018 1 1129 2000 97.1% chr1 - 148274103 148486535 212433 browser details YourSeq 1014 63 1134 2000 97.9% chr16 - 81071667 81072883 1217 browser details YourSeq 1003 88 1137 2000 97.9% chr17 - 84882919 84883972 1054 browser details YourSeq 999 63 1130 2000 98.5% chr16 - 80304394 80305489 1096 browser details YourSeq 999 51 1129 2000 97.8% chr13 - 75677018 75678188 1171 browser details YourSeq 999 82 1129 2000 97.9% chr10 + 45559189 45560280 1092 browser details YourSeq 998 1 1129 2000 96.3% chr17 + 69541164 69542282 1119 browser details YourSeq 996 45 1129 2000 98.0% chr6 - 18757495 18758636 1142 browser details YourSeq 992 63 1129 2000 98.3% chr14 - 42775516 42776612 1097 browser details YourSeq 987 82 1130 2000 98.0% chr7 + 138695194 138696237 1044 browser details YourSeq 985 63 1129 2000 97.9% chr11 - 81545481 81546581 1101 browser details YourSeq 984 63 1130 2000 97.9% chr13 - 117295788 117296886 1099 Note: The 2000 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% chr18 + 60538444 60540443 2000 browser details YourSeq 126 213 389 2000 90.4% chr2 + 60266427 60266611 185 browser details YourSeq 117 251 626 2000 84.6% chr13 - 44660875 44661242 368 browser details YourSeq 113 161 389 2000 91.9% chr5 + 124428158 124428488 331 browser details YourSeq 109 225 385 2000 92.4% chrX + 13060357 13060525 169 browser details YourSeq 102 188 390 2000 89.4% chr5 + 128959532 128959758 227 browser details YourSeq 100 177 390 2000 95.5% chr12 - 21382910 21383243 334 browser details YourSeq 97 265 389 2000 87.9% chr1 - 136994105 136994228 124 browser details YourSeq 95 213 389 2000 92.9% chr4 + 117102751 117102930 180 browser details YourSeq 94 1713 1862 2000 88.8% chr10 + 125034571 125034722 152 browser details YourSeq 93 265 390 2000 88.2% chr11 - 117051787 117051911 125 browser details YourSeq 91 199 389 2000 88.9% chr7 - 107045313 107045508 196 browser details YourSeq 91 265 391 2000 86.7% chr19 + 11940551 11940676 126 browser details YourSeq 90 207 387 2000 91.9% chr1 + 162543804 162543991 188 browser details YourSeq 89 267 390 2000 87.7% chr2 - 32048056 32048178 123 browser details YourSeq 89 178 386 2000 91.0% chr15 - 76641623 76642000 378 browser details YourSeq 88 214 369 2000 87.3% chr1 + 58960617 58960766 150 browser details YourSeq 87 267 390 2000 89.6% chr3 - 81280838 81280959 122 browser details YourSeq 87 272 386 2000 91.6% chr11 - 53426374 53426495 122 browser details YourSeq 86 212 357 2000 83.4% chr17 - 85918916 85919041 126 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: Dctn4 dynactin 4 [ Mus musculus (house mouse) ] Gene ID: 67665, updated on 24-Oct-2019 Gene summary Official Symbol Dctn4 provided by MGI Official Full Name dynactin 4 provided by MGI Primary source MGI:MGI:1914915 See related Ensembl:ENSMUSG00000024603 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 p62; 1110001K06Rik; 4930547K17Rik; C130039E17Rik Expression Ubiquitous expression in CNS E18 (RPKM 20.0), whole brain E14.5 (RPKM 18.4) and 28 other tissues See more Orthologs human all Genomic context Location: 18; 18 D3 See Dctn4 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (60526154..60558766) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (60685875..60718416) Chromosome 18 - NC_000084.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Dctn4 ENSMUSG00000024603 Description dynactin 4 [Source:MGI Symbol;Acc:MGI:1914915] Gene Synonyms 1110001K06Rik, 4930547K17Rik, C130039E17Rik, p62 Location Chromosome 18: 60,526,185-60,558,766 forward strand. GRCm38:CM001011.2 About this gene This gene has 8 transcripts (splice variants), 199 orthologues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Dctn4-201 ENSMUST00000025505.6 3738 460aa ENSMUSP00000025505.5 Protein coding CCDS37832 Q3TQY2 Q8CBY8 TSL:1 GENCODE basic APPRIS P2 Dctn4-204 ENSMUST00000223984.1 2948 467aa ENSMUSP00000153008.1 Protein coding - Q8CBY8 GENCODE basic APPRIS ALT1 Dctn4-202 ENSMUST00000223590.1 1530 48aa ENSMUSP00000153421.1 Protein coding - Q3TRR2 GENCODE basic Dctn4-205 ENSMUST00000224317.1 4331 No protein - Retained intron - - - Dctn4-203 ENSMUST00000223794.1 1724 No protein - Retained intron - - - Dctn4-208 ENSMUST00000225005.1 797 No protein - Retained intron - - - Dctn4-207 ENSMUST00000224778.1 691 No protein - Retained intron - - - Dctn4-206 ENSMUST00000224671.1 307 No protein - Retained intron - - - 52.58 kb Forward strand 60.52Mb 60.53Mb 60.54Mb 60.55Mb 60.56Mb Genes (Comprehensive set... Dctn4-204 >protein coding Rbm22-201 >protein coding Dctn4-201 >protein coding Rbm22-203 >retained intron Dctn4-202 >protein coding Dctn4-208 >retained intron Dctn4-206 >retained intron Dctn4-203 >retained intron Rbm22-205 >retained intron Dctn4-205 >retained intron Dctn4-207 >retained intron Contigs < AC135638.5 Regulatory Build 60.52Mb 60.53Mb 60.54Mb 60.55Mb 60.56Mb Reverse strand 52.58 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000025505 32.57 kb Forward strand Dctn4-201 >protein coding ENSMUSP00000025... Low complexity (Seg) Pfam Dynactin subunit 4 PANTHER Dynactin subunit 4 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 460 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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