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Mouse Ddx25 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ddx25 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ddx25 gene (NCBI Reference Sequence: NM_013932 ; Ensembl: ENSMUSG00000032101 ) is located on Mouse chromosome 9. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000034612). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ddx25 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-387H14 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 null mice display male infertility, arrest of spermatogenesis at step 8, abnormal Leydig cells, and increased germ cell apoptosis in males.

Exon 5 starts from about 21.69% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2156 bp, and the size of intron 6 for 3'-loxP site insertion: 1355 bp. The size of effective cKO region: ~803 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 5 6 7 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ddx25 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(7303bp) | A(26.58% 1941) | C(22.35% 1632) | T(28.71% 2097) | G(22.36% 1633)

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% chr9 - 35554860 35557859 3000 browser details YourSeq 76 2105 2208 3000 87.4% chr14 + 7833884 7833983 100 browser details YourSeq 73 2108 2208 3000 87.0% chr13 + 59511414 59511510 97 browser details YourSeq 72 2071 2209 3000 85.4% chr11 - 50781322 50781457 136 browser details YourSeq 68 2108 2208 3000 84.4% chr2 - 171025520 171025616 97 browser details YourSeq 68 2108 2208 3000 85.2% chr11 + 104511674 104511770 97 browser details YourSeq 67 2108 2205 3000 85.0% chr2 - 94041054 94041147 94 browser details YourSeq 67 2105 2208 3000 86.9% chr19 - 53995597 53995696 100 browser details YourSeq 67 2108 2208 3000 86.9% chr14 - 67694806 67694901 96 browser details YourSeq 66 2146 2240 3000 84.0% chr14 - 100469661 100469747 87 browser details YourSeq 65 2105 2208 3000 86.5% chr12 - 55917942 55918041 100 browser details YourSeq 65 2106 2208 3000 84.7% chr11 - 82906067 82906165 99 browser details YourSeq 65 2108 2204 3000 84.2% chr1 - 45930625 45930717 93 browser details YourSeq 64 2108 2208 3000 86.4% chr2 - 85039227 85039323 97 browser details YourSeq 64 2108 2208 3000 86.4% chr3 + 84648099 84648195 97 browser details YourSeq 64 2108 2208 3000 86.4% chr16 + 35331276 35331372 97 browser details YourSeq 63 2108 2208 3000 82.4% chr10 - 62932374 62932471 98 browser details YourSeq 62 2108 2208 3000 85.0% chr15 - 73208176 73208272 97 browser details YourSeq 62 2108 2208 3000 86.2% chr17 + 88259596 88259692 97 browser details YourSeq 61 2109 2208 3000 84.8% chr2 - 155037752 155037847 96

Note: The 3000 bp section upstream of Exon 5 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% chr9 - 35551057 35554056 3000 browser details YourSeq 151 2304 2476 3000 94.3% chr19 + 47228039 47228214 176 browser details YourSeq 150 2299 2473 3000 93.7% chr2 - 20285881 20286056 176 browser details YourSeq 149 2320 2478 3000 96.9% chr1 - 63500980 63501138 159 browser details YourSeq 146 2325 2476 3000 98.1% chr6 - 65151999 65152150 152 browser details YourSeq 145 2316 2476 3000 93.8% chr7 + 72339850 72340009 160 browser details YourSeq 144 2317 2476 3000 94.4% chr4 - 149776528 149776686 159 browser details YourSeq 144 2317 2476 3000 94.3% chr15 + 59101626 59101783 158 browser details YourSeq 143 2042 2458 3000 86.0% chr4 - 17385190 17385589 400 browser details YourSeq 143 2321 2476 3000 94.2% chr3 - 145355996 145356149 154 browser details YourSeq 143 2316 2476 3000 95.0% chr15 - 36940630 36940792 163 browser details YourSeq 142 2320 2476 3000 92.9% chrX - 22087934 22088087 154 browser details YourSeq 142 2315 2494 3000 92.3% chr11 + 75527803 75527982 180 browser details YourSeq 142 2317 2476 3000 95.0% chr11 + 12400913 12401084 172 browser details YourSeq 141 2311 2473 3000 93.8% chr2 - 107593537 107593954 418 browser details YourSeq 141 2316 2476 3000 96.1% chr5 + 122411321 122411485 165 browser details YourSeq 141 2317 2476 3000 92.5% chr12 + 111873216 111873373 158 browser details YourSeq 141 2320 2476 3000 93.0% chr10 + 50440966 50441120 155 browser details YourSeq 140 2316 2476 3000 92.4% chr4 - 11017858 11018016 159 browser details YourSeq 140 2316 2476 3000 93.8% chr10 + 30428175 30428343 169

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ddx25 DEAD (Asp-Glu-Ala-Asp) box polypeptide 25 [ Mus musculus (house mouse) ] Gene ID: 30959, updated on 10-Oct-2019

Gene summary

Official Symbol Ddx25 provided by MGI Official Full Name DEAD (Asp-Glu-Ala-Asp) box polypeptide 25 provided by MGI Primary source MGI:MGI:1353582 See related Ensembl:ENSMUSG00000032101 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 GRTH; AW047046 Expression Biased expression in testis adult (RPKM 116.1), CNS E18 (RPKM 19.3) and 5 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 A4 See Ddx25 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (35541848..35558470, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (35349433..35366055, complement)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 1 transcript

Gene: Ddx25 ENSMUSG00000032101

Description DEAD (Asp-Glu-Ala-Asp) box polypeptide 25 [Source:MGI Symbol;Acc:MGI:1353582] Gene Synonyms GRTH Location Chromosome 9: 35,536,647-35,558,528 reverse strand. GRCm38:CM001002.2 About this gene This gene has 1 transcript (splice variant), 139 orthologues, 39 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ddx25-201 ENSMUST00000034612.6 6934 484aa ENSMUSP00000034612.4 Protein coding CCDS52753 Q9QY15 TSL:1 GENCODE basic APPRIS P1

41.88 kb Forward strand 35.53Mb 35.54Mb 35.55Mb 35.56Mb Genes Pus3-203 >retained intron (Comprehensive set...

Pus3-201 >protein coding

Pus3-202 >protein coding

Contigs < AC118232.10 Genes (Comprehensive set... < Ddx25-201protein coding < Hyls1-201protein coding

< Hyls1-202retained intron

Regulatory Build

35.53Mb 35.54Mb 35.55Mb 35.56Mb Reverse strand 41.88 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000034612

< Ddx25-201protein coding

Reverse strand 21.88 kb

ENSMUSP00000034... 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 RNA helicase, DEAD-box type, Q motif Helicase, C-terminal

Helicase superfamily 1/2, ATP-binding domain PANTHER PTHR24031

PTHR24031:SF331 Gene3D 3.40.50.300 CDD cd18048 cd18787

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

Variant Legend inframe insertion synonymous variant

Scale bar 0 60 120 180 240 300 360 420 484

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

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