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Mouse Dppa3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Dppa3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dppa3 gene (NCBI Reference Sequence: NM_139218 ; Ensembl: ENSMUSG00000046323 ) is located on Mouse chromosome 6. 4 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 4 (Transcript: ENSMUST00000049644). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Dppa3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-68I7 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: Female mice homozygous for a disruption in this gene are infertile or have reduced fertility due to a failure in embryonic development at or before implantation.

Exon 2 starts from about 19.78% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1966 bp, and the size of intron 2 for 3'-loxP site insertion: 491 bp. The size of effective cKO region: ~728 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 2 3 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Dppa3 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(7223bp) | A(25.57% 1847) | C(21.85% 1578) | T(28.49% 2058) | G(24.09% 1740)

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% chr6 + 122625328 122628327 3000 browser details YourSeq 176 1099 1283 3000 97.9% chrX - 37626012 37626197 186 browser details YourSeq 150 774 1266 3000 89.8% chr14 + 34262561 34263193 633 browser details YourSeq 122 1126 1267 3000 93.0% chr14 + 30397921 30398062 142 browser details YourSeq 113 2696 2961 3000 90.3% chr15 - 53124396 53124659 264 browser details YourSeq 109 2686 2827 3000 93.0% chr2 - 70736914 70737408 495 browser details YourSeq 108 575 806 3000 94.4% chr10 - 3946046 3946278 233 browser details YourSeq 107 2696 2827 3000 94.2% chr11 + 117805454 117805606 153 browser details YourSeq 104 2696 2828 3000 94.1% chr18 + 35856924 35958111 101188 browser details YourSeq 103 2696 2827 3000 91.9% chr6 + 125497689 125497838 150 browser details YourSeq 103 2696 2828 3000 94.0% chr10 + 127115762 127115907 146 browser details YourSeq 102 2696 2827 3000 93.3% chr2 - 144582696 144582842 147 browser details YourSeq 102 2696 2821 3000 94.0% chr5 + 145075865 145076011 147 browser details YourSeq 102 1120 1262 3000 86.6% chr19 + 53883293 53883430 138 browser details YourSeq 101 2696 2829 3000 93.2% chr1 + 156116053 156116207 155 browser details YourSeq 100 2696 2819 3000 94.6% chr11 + 4962356 4962494 139 browser details YourSeq 100 2696 2819 3000 93.9% chr10 + 7020300 7020440 141 browser details YourSeq 99 678 810 3000 91.6% chr1 - 95173521 95173658 138 browser details YourSeq 98 2696 2818 3000 93.8% chr5 - 121898519 121898663 145 browser details YourSeq 97 2699 2819 3000 95.3% chr5 - 92045833 92045966 134

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% chr6 + 122629056 122632055 3000 browser details YourSeq 572 2185 2766 3000 98.8% chr4 + 144846232 144846812 581 browser details YourSeq 572 2185 2766 3000 98.8% chr14 + 8071268 8071848 581 browser details YourSeq 570 2185 2766 3000 98.7% chr7 - 58163277 58163857 581 browser details YourSeq 570 2185 2766 3000 98.7% chr14 - 53067960 53068540 581 browser details YourSeq 570 2185 2766 3000 98.7% chr5 + 138071068 138071648 581 browser details YourSeq 570 2185 2766 3000 98.7% chr1 + 10979518 10980098 581 browser details YourSeq 568 2185 2766 3000 98.5% chr9 - 121180663 121181243 581 browser details YourSeq 568 2185 2766 3000 98.5% chr7 - 130347498 130348078 581 browser details YourSeq 568 2185 2766 3000 98.5% chr7 - 89860161 89860741 581 browser details YourSeq 568 2185 2766 3000 98.5% chr7 - 15808313 15808893 581 browser details YourSeq 568 2185 2766 3000 98.5% chr2 - 114183151 114183731 581 browser details YourSeq 568 2185 2766 3000 98.5% chr2 - 112322043 112322623 581 browser details YourSeq 568 2185 2766 3000 98.5% chr2 - 57168711 57169291 581 browser details YourSeq 568 2185 2766 3000 98.5% chr1_GL456211_random - 187100 187680 581 browser details YourSeq 568 2185 2766 3000 98.5% chr16 - 57203260 57203840 581 browser details YourSeq 568 2185 2766 3000 98.5% chr16 - 8474914 8475494 581 browser details YourSeq 568 2185 2766 3000 98.5% chr11 - 94625200 94625780 581 browser details YourSeq 568 2185 2766 3000 98.5% chr10 - 79208371 79208951 581 browser details YourSeq 568 2185 2766 3000 98.5% chrX + 109268669 109269249 581

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Dppa3 developmental pluripotency-associated 3 [ Mus musculus (house mouse) ] Gene ID: 73708, updated on 10-Sep-2019

Gene summary

Official Symbol Dppa3 provided by MGI Official Full Name developmental pluripotency-associated 3 provided by MGI Primary source MGI:MGI:1920958 See related Ensembl:ENSMUSG00000046323 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as PCG7; PGC7; Stella; 2410075G02Rik Expression Low expression observed in reference dataset See more Orthologs human all

Genomic context

Location: 6; 6 F1 See Dppa3 in Genome Data Viewer

Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (122626424..122630271)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (122576442..122580289)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 2 transcripts

Gene: Dppa3 ENSMUSG00000046323

Description developmental pluripotency-associated 3 [Source:MGI Symbol;Acc:MGI:1920958] Gene Synonyms 2410075G02Rik, PGC7, stella Location Chromosome 6: 122,626,410-122,630,272 forward strand. GRCm38:CM000999.2 About this gene This gene has 2 transcripts (splice variants), 79 orthologues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dppa3-201 ENSMUST00000049644.8 833 150aa ENSMUSP00000062832.2 Protein coding CCDS20500 Q8QZY3 TSL:1 GENCODE basic APPRIS P1

Dppa3-202 ENSMUST00000123429.1 691 141aa ENSMUSP00000115252.1 Protein coding - K4DID3 CDS 5' incomplete TSL:5

23.86 kb Forward strand 122.62Mb 122.63Mb 122.64Mb Genes (Comprehensive set... Gm26168-201 >misc RNDAppa3-202 >protein coding

Dppa3-201 >protein coding

Contigs AC158651.12 > Regulatory Build

122.62Mb 122.63Mb 122.64Mb Reverse strand 23.86 kb

Regulation Legend CTCF Enhancer Open Chromatin 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: ENSMUST00000049644

3.86 kb Forward strand

Dppa3-201 >protein coding

ENSMUSP00000062... MobiDB lite Low complexity (Seg) Pfam Developmental pluripotency-associated protein 3 PANTHER Developmental pluripotency-associated protein 3

PTHR31577:SF2

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

Variant Legend stop lost missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 150

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

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