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Mouse Tdrd3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tdrd3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tdrd3 gene (NCBI Reference Sequence: NM_001253755 ; Ensembl: ENSMUSG00000022019 ) is located on Mouse chromosome 14. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000169504). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tdrd3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-350G18 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: Homozygous mice for a hypomorphic allele show increased genomic instability, and increased c-Myc/Igh translocation and R loop formation in B cells.

Exon 4 starts from about 8.99% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 13253 bp, and the size of intron 4 for 3'-loxP site insertion: 2462 bp. The size of effective cKO region: ~661 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 4 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tdrd3 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(7161bp) | A(26.31% 1884) | C(17.75% 1271) | T(36.45% 2610) | G(19.49% 1396)

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% chr14 + 87468827 87471826 3000 browser details YourSeq 81 535 839 3000 77.1% chr10 - 16515840 16516050 211 browser details YourSeq 75 1800 2046 3000 85.9% chr16 - 64672946 64673184 239 browser details YourSeq 71 1828 2022 3000 88.3% chr13 - 60310481 60310678 198 browser details YourSeq 71 535 847 3000 75.0% chr1 - 11928734 11928952 219 browser details YourSeq 70 535 953 3000 71.0% chr6 + 69885538 69885862 325 browser details YourSeq 69 1900 2034 3000 92.7% chr2 - 31343566 31343708 143 browser details YourSeq 68 1463 2032 3000 68.2% chr15 + 67244744 67244897 154 browser details YourSeq 64 535 733 3000 82.2% chr1 + 142018325 142018518 194 browser details YourSeq 63 1927 2041 3000 89.9% chr9 - 94424219 94424334 116 browser details YourSeq 63 1928 2045 3000 91.0% chr17 - 52022969 52023086 118 browser details YourSeq 60 550 736 3000 90.7% chr16 - 47045969 47046156 188 browser details YourSeq 59 1897 2034 3000 87.4% chr10 + 128029409 128029557 149 browser details YourSeq 58 1917 2034 3000 90.3% chr9 + 104531448 104531577 130 browser details YourSeq 56 535 847 3000 72.6% chr14 - 53406340 53406559 220 browser details YourSeq 56 1913 2031 3000 92.6% chr1 - 85996739 85996857 119 browser details YourSeq 56 1901 2042 3000 71.5% chr7 + 79295199 79307979 12781 browser details YourSeq 56 918 1134 3000 93.9% chr18 + 73632154 73632372 219 browser details YourSeq 56 1935 2079 3000 79.5% chr16 + 14043710 14043840 131 browser details YourSeq 56 1761 2041 3000 76.0% chr14 + 33850741 33850999 259

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% chr14 + 87472488 87475487 3000 browser details YourSeq 222 1194 1484 3000 91.5% chr8 - 103192738 103193085 348 browser details YourSeq 57 1749 1889 3000 89.4% chr11 + 72106035 72106425 391 browser details YourSeq 53 1772 1875 3000 93.4% chr11 + 68935723 68935864 142 browser details YourSeq 50 1734 1810 3000 93.2% chr1 - 59525780 59525873 94 browser details YourSeq 48 1751 1811 3000 90.0% chr12 - 83230755 83230825 71 browser details YourSeq 46 1753 1811 3000 89.7% chr9 + 50532882 50532950 69 browser details YourSeq 45 1751 1810 3000 89.5% chr10 + 89107437 89107506 70 browser details YourSeq 45 1764 1816 3000 88.5% chr10 + 25259040 25259091 52 browser details YourSeq 44 1762 1809 3000 95.9% chr11 - 100389258 100389305 48 browser details YourSeq 44 1722 1810 3000 71.7% chr10 - 71180295 71180364 70 browser details YourSeq 44 1761 1818 3000 85.8% chrX + 98965511 98965567 57 browser details YourSeq 43 1762 1810 3000 93.9% chr11 - 120758947 120758995 49 browser details YourSeq 43 1762 1810 3000 93.9% chr2 + 167083713 167083761 49 browser details YourSeq 43 1764 1819 3000 86.8% chr11 + 105087230 105087284 55 browser details YourSeq 42 1762 1810 3000 95.7% chr9 - 22166931 22166979 49 browser details YourSeq 42 1762 1809 3000 89.4% chr1 - 155291084 155291130 47 browser details YourSeq 41 1762 1810 3000 91.9% chr8 - 106942988 106943036 49 browser details YourSeq 41 1762 1810 3000 91.9% chr12 - 80987868 80987916 49 browser details YourSeq 41 1762 1810 3000 91.9% chr11 - 115191133 115191181 49

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tdrd3 tudor domain containing 3 [ Mus musculus (house mouse) ] Gene ID: 219249, updated on 12-Aug-2019

Gene summary

Official Symbol Tdrd3 provided by MGI Official Full Name tudor domain containing 3 provided by MGI Primary source MGI:MGI:2444023 See related Ensembl:ENSMUSG00000022019 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 6720468N18; 4732418C03Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 1.6), limb E14.5 (RPKM 1.5) and 28 other tissues See more Orthologs human all

Genomic context

Location: 14; 14 E1 See Tdrd3 in Genome Data Viewer

Exon count: 19

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (87416178..87545508)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (87816390..87945315)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 6 transcripts

Gene: Tdrd3 ENSMUSG00000022019

Description tudor domain containing 3 [Source:MGI Symbol;Acc:MGI:2444023] Gene Synonyms 4732418C03Rik Location Chromosome 14: 87,416,639-87,545,504 forward strand. GRCm38:CM001007.2 About this gene This gene has 6 transcripts (splice variants), 247 orthologues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tdrd3-203 ENSMUST00000169504.7 5280 716aa ENSMUSP00000131542.1 Protein coding CCDS56972 Q91W18 TSL:1 GENCODE basic

Tdrd3-202 ENSMUST00000168275.8 2657 743aa ENSMUSP00000129019.1 Protein coding CCDS27305 Q91W18 TSL:1 GENCODE basic APPRIS P1

Tdrd3-205 ENSMUST00000170865.7 2530 737aa ENSMUSP00000126189.1 Protein coding - Q91W18 TSL:1 GENCODE basic

Tdrd3-204 ENSMUST00000170712.7 636 212aa ENSMUSP00000126212.1 Protein coding - F6ZI20 CDS 5' and 3' incomplete TSL:3

Tdrd3-201 ENSMUST00000163714.1 849 No protein - lncRNA - - TSL:3

Tdrd3-206 ENSMUST00000227304.1 669 No protein - lncRNA - - -

148.87 kb Forward strand 87.42Mb 87.44Mb 87.46Mb 87.48Mb 87.50Mb 87.52Mb 87.54Mb Genes (Comprehensive set... Tdrd3-202 >protein coding

Tdrd3-203 >protein coding Tdrd3-206 >lncRNA

Tdrd3-205 >protein coding

Gm49199-201 >TEC Tdrd3-204 >protein coding Gm49197-201 >TEC

Tdrd3-201 >lncRNA

Contigs AC154562.2 > < CT010453.10 Genes < Gm33121-201processed pseudogene (Comprehensive set...

Regulatory Build

87.42Mb 87.44Mb 87.46Mb 87.48Mb 87.50Mb 87.52Mb 87.54Mb Reverse strand 148.87 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript pseudogene

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Transcript: ENSMUST00000169504

98.13 kb Forward strand

Tdrd3-203 >protein coding

ENSMUSP00000131... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily UBA-like superfamily SSF63748

SMART RecQ mediated genome instability protein, DUF1767 Tudor domain

Ubiquitin-associated domain Pfam RecQ mediated genome instability protein, N-terminal Survival motor neuron

Ubiquitin-associated domain PROSITE profiles Ubiquitin-associated domain Tudor domain

PANTHER PTHR13681

PTHR13681:SF24 Gene3D RecQ mediated genome instability protein, N-terminal, subdomain 2 2.30.30.140

1.10.8.10 CDD cd04488 Tudor domain-containing protein 3, UBA domain cd04508

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 716

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

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