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Mouse Ddx3y Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ddx3y conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ddx3y gene (NCBI Reference Sequence: NM_012008 ; Ensembl: ENSMUSG00000069045 ) is located on Mouse chromosome Y. 17 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 17 (Transcript: ENSMUST00000091190). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ddx3y gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-208N6 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 2 starts from about 2.33% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 2096 bp, and the size of intron 3 for 3'-loxP site insertion: 2461 bp. The size of effective cKO region: ~1982 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 2 3 17 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Ddx3y 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(8482bp) | A(30.46% 2584) | C(15.96% 1354) | T(33.8% 2867) | G(19.77% 1677)

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% chrY - 1284633 1287632 3000 browser details YourSeq 92 1762 2251 3000 74.8% chr18 - 73984511 73984743 233 browser details YourSeq 86 1805 2299 3000 89.9% chr4 + 129425391 129425944 554 browser details YourSeq 85 2145 2300 3000 85.8% chr18 - 34573161 34573317 157 browser details YourSeq 82 1756 2285 3000 73.1% chr9 + 109554801 109555060 260 browser details YourSeq 80 1756 2227 3000 76.1% chr11 - 20087456 20087778 323 browser details YourSeq 76 2158 2305 3000 86.6% chr2 - 130561280 130561428 149 browser details YourSeq 76 2145 2285 3000 87.5% chr4 + 54810778 54810914 137 browser details YourSeq 72 2165 2305 3000 84.7% chr17 - 71568232 71568371 140 browser details YourSeq 72 1756 2253 3000 72.8% chr11 - 86216097 86216373 277 browser details YourSeq 71 1756 2285 3000 73.2% chr6 + 128419513 128419825 313 browser details YourSeq 71 2155 2285 3000 88.6% chr4 + 149271709 149271838 130 browser details YourSeq 70 1805 2255 3000 71.0% chr9 - 107643112 107643300 189 browser details YourSeq 70 1756 2255 3000 71.5% chr7 + 66515720 66515962 243 browser details YourSeq 69 2153 2255 3000 82.4% chr4 - 124864971 124865063 93 browser details YourSeq 68 2160 2282 3000 90.5% chr1 - 135904826 135904948 123 browser details YourSeq 68 1805 2285 3000 91.4% chr1 + 86710261 86710816 556 browser details YourSeq 65 2165 2497 3000 70.4% chr11 - 5177781 5177891 111 browser details YourSeq 62 2197 2305 3000 88.8% chr1 - 86573463 86927586 354124 browser details YourSeq 61 2165 2284 3000 85.6% chrX + 138031674 138031787 114

Note: The 3000 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 3000 1 3000 3000 100.0% chrY - 1279651 1282650 3000 browser details YourSeq 145 1152 1338 3000 89.2% chrX + 91292968 91293166 199 browser details YourSeq 143 1150 1331 3000 92.0% chr7 + 3236395 3605205 368811 browser details YourSeq 143 1144 1332 3000 90.4% chr6 + 87968378 88089516 121139 browser details YourSeq 140 1151 1338 3000 87.7% chrX - 92046545 92046744 200 browser details YourSeq 135 1150 1332 3000 89.1% chr2 - 152419740 152419931 192 browser details YourSeq 132 1148 1361 3000 90.7% chr14 - 86561470 86561887 418 browser details YourSeq 132 1145 1348 3000 86.7% chr1 - 116054710 116054916 207 browser details YourSeq 132 1160 1334 3000 89.3% chr4 + 135813120 135980056 166937 browser details YourSeq 132 1152 1333 3000 90.2% chr11 + 87419593 87419776 184 browser details YourSeq 130 1141 1333 3000 86.0% chr10 + 60241830 60242021 192 browser details YourSeq 129 33 1307 3000 86.5% chr4 - 122967680 123280296 312617 browser details YourSeq 126 1151 1331 3000 90.4% chr18 + 3236063 3236246 184 browser details YourSeq 126 1150 1331 3000 89.4% chr17 + 70797271 70797455 185 browser details YourSeq 123 1154 1335 3000 90.2% chr14 + 54525658 54526217 560 browser details YourSeq 122 1150 1331 3000 88.2% chr16 + 97796005 97796191 187 browser details YourSeq 121 1148 1332 3000 89.5% chr6 - 70877603 70877784 182 browser details YourSeq 121 1150 1332 3000 89.6% chr4 - 129885212 129885401 190 browser details YourSeq 121 1153 1338 3000 91.1% chr12 - 110645656 111017872 372217 browser details YourSeq 119 1169 1328 3000 89.5% chr15 + 84876624 84876790 167

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ddx3y DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked [ Mus musculus (house mouse) ] Gene ID: 26900, updated on 10-Oct-2019

Gene summary

Official Symbol Ddx3y provided by MGI Official Full Name DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked provided by MGI Primary source MGI:MGI:1349406 See related Ensembl:ENSMUSG00000069045 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 Dby; D1Pas1-rs1; 8030469F12Rik Expression Broad expression in CNS E18 (RPKM 5.2), liver E14.5 (RPKM 4.5) and 17 other tissues See more

Genomic context

Location: Y; Y A1 See Ddx3y in Genome Data Viewer Exon count: 18

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) Y NC_000087.7 (1260715..1286628, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) Y NC_000087.6 (597158..623056, complement)

Chromosome Y - NC_000087.7

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Transcript information: This gene has 4 transcripts

Gene: Ddx3y ENSMUSG00000069045

Description DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked [Source:MGI Symbol;Acc:MGI:1349406] Gene Synonyms 8030469F12Rik, D1Pas1-rs1, Dby Location Chromosome Y: 1,260,771-1,286,629 reverse strand. GRCm38:CM001014.2 About this gene This gene has 4 transcripts (splice variants), 196 orthologues, 39 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ddx3y- ENSMUST00000091190.11 4600 658aa ENSMUSP00000088729.5 Protein coding CCDS30543 Q62095 TSL:1 201 GENCODE basic APPRIS P1

Ddx3y- ENSMUST00000188484.1 3031 41aa ENSMUSP00000140361.1 Nonsense mediated - A0A087WQV7 TSL:1 204 decay

Ddx3y- ENSMUST00000188182.6 3072 No - Retained intron - - TSL:1 203 protein

Ddx3y- ENSMUST00000187596.1 664 No - Retained intron - - TSL:NA 202 protein

45.86 kb Forward strand 1.26Mb 1.27Mb 1.28Mb 1.29Mb Contigs AC145393.4 >

Genes (Comprehensive set... < Ddx3y-201protein coding

< Ddx3y-203retained intron < Gm4017-201processed pseudoge

< Ddx3y-204nonsense mediated decay

< Ddx3y-202retained intron

Regulatory Build

1.26Mb 1.27Mb 1.28Mb 1.29Mb Reverse strand 45.86 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

pseudogene processed transcript

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Transcript: ENSMUST00000091190

< Ddx3y-201protein coding

Reverse strand 25.86 kb

ENSMUSP00000088... MobiDB lite Low complexity (Seg) Superfamily P-loop containing nucleoside triphosphate hydrolase SMART Helicase superfamily 1/2, ATP-binding domain Helicase, C-terminal

Pfam DEAD/DEAH box helicase domain Helicase, C-terminal

PROSITE profiles Helicase superfamily 1/2, ATP-binding domain

RNA helicase, DEAD-box type, Q motif Helicase, C-terminal PROSITE patterns ATP-dependent RNA helicase DEAD-box, conserved site

PANTHER PTHR24031:SF548

PTHR24031 Gene3D 3.40.50.300 CDD cd18051 cd18787

Scale bar 0 60 120 180 240 300 360 420 480 540 658

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

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