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Mouse Dctn2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Dctn2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dctn2 gene (NCBI Reference Sequence: NM_027151 ; Ensembl: ENSMUSG00000025410 ) is located on Mouse chromosome 10. 14 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000026479). Exon 6~13 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Dctn2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-257L23 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 6~13 is not frameshift exon, and covers 62.94% of the coding region. The size of intron 5 for 5'-loxP site insertion: 461 bp, and the size of intron 13 for 3'-loxP site insertion: 2992 bp. The size of effective cKO region: ~2505 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 3 4 5 6 7 8 9 10 11 12 13 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Dctn2 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8985bp) | A(22.78% 2047) | C(24.05% 2161) | T(27.05% 2430) | G(26.12% 2347)

Note: The sequence of homologous arms and cKO region 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.

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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% chr10 + 127273155 127276154 3000 browser details YourSeq 146 2058 2409 3000 91.0% chr17 + 26159490 26159862 373 browser details YourSeq 130 2075 2359 3000 85.1% chr6 + 120099064 120099310 247 browser details YourSeq 125 2010 2199 3000 83.4% chr4 + 98905539 98905713 175 browser details YourSeq 123 2048 2200 3000 88.3% chr6 + 116925511 116925659 149 browser details YourSeq 120 2048 2200 3000 90.5% chr17 - 17852478 17852630 153 browser details YourSeq 120 2076 2382 3000 90.5% chr3 + 87213135 87213443 309 browser details YourSeq 119 2050 2206 3000 90.0% chr5 + 25214642 25214800 159 browser details YourSeq 118 2037 2186 3000 89.8% chr10 + 81507501 81507660 160 browser details YourSeq 117 2050 2200 3000 89.9% chr13 + 66962427 66962579 153 browser details YourSeq 115 2019 2186 3000 83.4% chr13 + 63195976 63196120 145 browser details YourSeq 114 2049 2298 3000 82.9% chr11 - 100530691 100530935 245 browser details YourSeq 114 2048 2186 3000 92.5% chr11 - 68112922 68113060 139 browser details YourSeq 113 2059 2222 3000 89.2% chr1 - 182253153 182253324 172 browser details YourSeq 111 2050 2186 3000 91.8% chr8 - 110977570 110977710 141 browser details YourSeq 110 2049 2186 3000 87.2% chr14 - 121411617 121411749 133 browser details YourSeq 108 2075 2210 3000 91.7% chr10 - 78494222 78494496 275 browser details YourSeq 108 2049 2188 3000 87.2% chr1 - 144968836 144968972 137 browser details YourSeq 107 2056 2299 3000 90.9% chr3 - 51323802 51324141 340 browser details YourSeq 107 2075 2210 3000 92.1% chr2 + 165808516 165808651 136

Note: The 3000 bp section upstream of Exon 6 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% chr10 + 127278660 127281659 3000 browser details YourSeq 183 786 1178 3000 91.5% chr9 - 72678139 72678602 464 browser details YourSeq 181 819 1183 3000 89.0% chr2 + 129797533 129797812 280 browser details YourSeq 172 629 1185 3000 89.7% chr1 - 64548474 64548999 526 browser details YourSeq 163 1028 1286 3000 93.1% chr7 - 79840298 79840557 260 browser details YourSeq 162 807 1188 3000 87.0% chr2 + 17983702 17984001 300 browser details YourSeq 145 1025 1187 3000 93.3% chr16 - 30516626 30516787 162 browser details YourSeq 144 2190 2670 3000 83.5% chr11 + 80077619 80077869 251 browser details YourSeq 140 1028 1178 3000 96.7% chr5 - 146220087 146220247 161 browser details YourSeq 136 1025 1170 3000 96.6% chr2 - 71139051 71139196 146 browser details YourSeq 134 1028 1196 3000 95.3% chr17 + 26236530 26236878 349 browser details YourSeq 130 1029 1166 3000 97.2% chr17 - 33295119 33295256 138 browser details YourSeq 128 1029 1164 3000 97.1% chr13 + 64302341 64302476 136 browser details YourSeq 115 796 1130 3000 84.1% chr17 - 28941666 28941946 281 browser details YourSeq 113 2202 2655 3000 77.5% chr1 - 179698055 179698398 344 browser details YourSeq 109 1036 1157 3000 95.1% chr10 - 28928687 28928811 125 browser details YourSeq 109 333 856 3000 79.0% chr11 + 96494086 96494302 217 browser details YourSeq 109 1044 1157 3000 98.3% chr11 + 78090476 78090595 120 browser details YourSeq 109 807 1130 3000 82.4% chr11 + 53340164 53340369 206 browser details YourSeq 107 1717 2246 3000 75.4% chr5 - 142913941 142914275 335

Note: The 3000 bp section downstream of Exon 13 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Dctn2 dynactin 2 [ Mus musculus (house mouse) ] Gene ID: 69654, updated on 12-Aug-2019

Gene summary

Official Symbol Dctn2 provided by MGI Official Full Name dynactin 2 provided by MGI Primary source MGI:MGI:107733 See related Ensembl:ENSMUSG00000025410 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 p50; RBP50; C87049; DCTN-50; GMP23-48K; 2310042E05Rik; C130077D06Rik Expression Ubiquitous expression in testis adult (RPKM 150.2), adrenal adult (RPKM 99.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 10 D3; 10 74.5 cM See Dctn2 in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (127266262..127281959)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (126703318..126719015)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 5 transcripts

Gene: Dctn2 ENSMUSG00000025410

Description dynactin 2 [Source:MGI Symbol;Acc:MGI:107733] Gene Synonyms 2310042E05Rik, C130077D06Rik, DCTN-50, RBP50, p50 Location Chromosome 10: 127,266,368-127,281,950 forward strand. GRCm38:CM001003.2 About this gene This gene has 5 transcripts (splice variants), 198 orthologues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dctn2-201 ENSMUST00000026479.10 1784 402aa ENSMUSP00000026479.9 Protein coding CCDS24234 Q3TPZ5 Q99KJ8 TSL:1 GENCODE basic APPRIS P1

Dctn2-202 ENSMUST00000217692.1 2557 No protein - Retained intron - - TSL:1

Dctn2-203 ENSMUST00000218556.1 882 No protein - Retained intron - - TSL:3

Dctn2-204 ENSMUST00000218752.1 820 No protein - Retained intron - - TSL:2

Dctn2-205 ENSMUST00000220418.1 521 No protein - Retained intron - - TSL:3

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35.58 kb Forward strand 127.26Mb 127.27Mb 127.28Mb 127.29Mb Genes (Comprehensive set... Dctn2-202 >retained intron Dctn2-205 >retained intron Ddit3-202 >retained intron

Dctn2-201 >protein coding Ddit3-201 >protein coding

Dctn2-203 >retained intron Ddit3-203 >protein coding

Dctn2-204 >retained intron Ddit3-204 >protein coding

Gm20492-201 >lncRNA

Contigs < AC144852.3 Genes < Kif5a-201protein coding < Mbd6-202protein coding (Comprehensive set...

< Kif5a-202protein coding < Mbd6-201protein coding

< Kif5a-203nonsense mediated decay < Mbd6-203nonsense mediated decay

< Mbd6-204nonsense mediated decay

< Mbd6-205protein coding < Gm4189-202lncRNA

< Mbd6-207protein coding

< Mbd6-206protein coding

< Gm4189-201retained intron

Regulatory Build

127.26Mb 127.27Mb 127.28Mb 127.29Mb Reverse strand 35.58 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000026479

15.56 kb Forward strand

Dctn2-201 >protein coding

ENSMUSP00000026... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Dynamitin PANTHER Dynamitin

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 402

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

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