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Mouse Dctn3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Dctn3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dctn3 gene (NCBI Reference Sequence: NM_016890 ; Ensembl: ENSMUSG00000028447 ) is located on Mouse chromosome 4. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000030158). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Dctn3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-32N21 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note:

Exon 4 starts from about 48.21% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2396 bp, and the size of intron 5 for 3'-loxP site insertion: 940 bp. The size of effective cKO region: ~1545 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 5 6 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Dctn3 Homology arm cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region 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 GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8045bp) | A(22.16% 1783) | C(26.55% 2136) | T(25.61% 2060) | G(25.68% 2066)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr4 - 41717691 41720690 3000 browser details YourSeq 86 1388 1642 3000 85.8% chr5 - 90853978 90854246 269 browser details YourSeq 69 1366 1641 3000 88.0% chr4 + 135403138 135403587 450 browser details YourSeq 69 1297 1642 3000 91.6% chr11 + 35323256 35323605 350 browser details YourSeq 66 1402 1642 3000 88.4% chr10 - 113731817 113732068 252 browser details YourSeq 66 1402 1640 3000 88.4% chr11 + 6559186 6559433 248 browser details YourSeq 65 1677 2140 3000 93.4% chr10 - 95845672 95846287 616 browser details YourSeq 64 1654 1880 3000 78.7% chr11 + 4934073 4934268 196 browser details YourSeq 63 1652 1885 3000 78.4% chr12 - 82206749 82206948 200 browser details YourSeq 57 1655 1880 3000 78.5% chr14 - 12668730 12668924 195 browser details YourSeq 57 1402 1641 3000 90.2% chr9 + 17785468 17785718 251 browser details YourSeq 56 1664 1847 3000 91.2% chr13 + 73976101 73976290 190 browser details YourSeq 54 1658 1878 3000 78.7% chr1 + 156613589 156613781 193 browser details YourSeq 53 1749 1838 3000 80.0% chr6 - 142799490 142799580 91 browser details YourSeq 52 1757 1880 3000 79.8% chr2 - 174711907 174712228 322 browser details YourSeq 51 1800 1878 3000 93.5% chr11 - 97754214 97754292 79 browser details YourSeq 50 1799 1878 3000 86.7% chr19 + 54239504 54239581 78 browser details YourSeq 50 1752 1879 3000 94.8% chr11 + 36130715 36130845 131 browser details YourSeq 48 1811 1875 3000 89.1% chr1 - 151797573 151797636 64 browser details YourSeq 47 1447 1641 3000 91.3% chr12 + 59094265 59094468 204

Note: The 3000 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 3000 1 3000 3000 100.0% chr4 - 41713146 41716145 3000 browser details YourSeq 141 1840 2003 3000 93.8% chr1 - 38188961 38189129 169 browser details YourSeq 139 1853 2003 3000 96.0% chr17 - 71080236 71080385 150 browser details YourSeq 138 1853 2003 3000 93.9% chr5 + 25549881 25550028 148 browser details YourSeq 136 1866 2003 3000 99.3% chr9 - 65931146 65931283 138 browser details YourSeq 136 1853 1998 3000 95.2% chr10 - 72675012 72675156 145 browser details YourSeq 135 1853 2001 3000 93.2% chr6 - 57649194 57649339 146 browser details YourSeq 134 1867 2005 3000 98.6% chr15 - 7164181 7164320 140 browser details YourSeq 134 1853 2002 3000 93.3% chr10 + 77170131 77170279 149 browser details YourSeq 133 1853 1997 3000 95.1% chr7 - 82748551 82748693 143 browser details YourSeq 133 1853 2003 3000 92.6% chr1 - 135176881 135177028 148 browser details YourSeq 133 1853 2001 3000 93.3% chr5 + 115801146 115801293 148 browser details YourSeq 133 1857 2003 3000 95.1% chr4 + 87650072 87650216 145 browser details YourSeq 133 1855 2003 3000 95.9% chr11 + 54567411 54567561 151 browser details YourSeq 132 1856 2003 3000 92.5% chr10 - 88155438 88155583 146 browser details YourSeq 131 1853 2003 3000 91.3% chr5 - 130160909 130161057 149 browser details YourSeq 131 1853 2003 3000 90.6% chr2 - 87462940 87463087 148 browser details YourSeq 131 1853 2003 3000 91.6% chr11 - 93900228 93900372 145 browser details YourSeq 131 1853 1997 3000 93.8% chr10 - 73277296 73277439 144 browser details YourSeq 131 1853 2003 3000 90.4% chr1 - 36536786 36536930 145

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Dctn3 dynactin 3 [ Mus musculus (house mouse) ] Gene ID: 53598, updated on 12-Aug-2019

Gene summary

Official Symbol Dctn3 provided by MGI Official Full Name dynactin 3 provided by MGI Primary source MGI:MGI:1859251 See related Ensembl:ENSMUSG00000028447 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 p24 Expression Ubiquitous expression in CNS E18 (RPKM 56.9), CNS E14 (RPKM 43.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 A5 See Dctn3 in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (41714798..41723163, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (41661830..41670195, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 3 transcripts

Gene: Dctn3 ENSMUSG00000028447

Description dynactin 3 [Source:MGI Symbol;Acc:MGI:1859251] Gene Synonyms p24 Location Chromosome 4: 41,714,798-41,723,170 reverse strand. GRCm38:CM000997.2 About this gene This gene has 3 transcripts (splice variants), 197 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dctn3-201 ENSMUST00000030158.10 973 186aa ENSMUSP00000030158.4 Protein coding CCDS18069 Q9Z0Y1 TSL:1 GENCODE basic APPRIS P1

Dctn3-203 ENSMUST00000171641.1 885 159aa ENSMUSP00000130988.1 Protein coding CCDS51143 E9Q919 TSL:3 GENCODE basic

Dctn3-202 ENSMUST00000147120.1 344 No protein - lncRNA - - TSL:2

28.37 kb Forward strand 41.71Mb 41.72Mb 41.73Mb Genes Il11ra1-203 >protein coding (Comprehensive set...

Contigs AL807796.17 > Genes (Comprehensive set... < Rpp25l-201protein coding < Dctn3-202lncRNA < Arid3c-203protein coding

< Dctn3-203protein coding < Arid3c-202protein coding

< Dctn3-201protein coding < Arid3c-201protein coding

Regulatory Build

41.71Mb 41.72Mb 41.73Mb Reverse strand 28.37 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000030158

< Dctn3-201protein coding

Reverse strand 8.37 kb

ENSMUSP00000030... Pfam Dynactin subunit 3 PANTHER Dynactin subunit 3

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend

missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 186

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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