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Mouse Pde4dip Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Pde4dip conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Pde4dip gene (NCBI Reference Sequence: NM_001289701 ; Ensembl: ENSMUSG00000038170 ) is located on Mouse chromosome 3. 47 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 47 (Transcript: ENSMUST00000090750). Exon 18~19 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pde4dip gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-299A21 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: Mice homozygous for a null allele exhibit partial (in utero or perinatal) lethality, hyperactivity, and increased vertical activity.

Exon 18 starts from about 31.68% of the coding region. The knockout of Exon 18~19 will result in frameshift of the gene. The size of intron 17 for 5'-loxP site insertion: 3873 bp, and the size of intron 19 for 3'-loxP site insertion: 753 bp. The size of effective cKO region: ~919 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 18 19 20 47 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pde4dip 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(7419bp) | A(24.13% 1790) | C(23.33% 1731) | T(29.44% 2184) | G(23.1% 1714)

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% chr3 - 97748186 97751185 3000 browser details YourSeq 46 2072 2119 3000 98.0% chr3 - 97749790 97749837 48 browser details YourSeq 46 1349 1396 3000 98.0% chr3 - 97749067 97749114 48 browser details YourSeq 42 1622 1691 3000 88.5% chrX + 92654111 92654181 71 browser details YourSeq 33 76 120 3000 81.9% chr13 + 12538758 12538801 44 browser details YourSeq 32 1066 1116 3000 78.1% chr7 - 39186366 39186411 46 browser details YourSeq 32 1046 1092 3000 81.9% chr3 - 103255210 103255255 46 browser details YourSeq 32 2107 2142 3000 94.5% chr18 + 59479203 59479238 36 browser details YourSeq 31 1235 1290 3000 97.2% chr6 - 134546070 134546127 58 browser details YourSeq 31 253 311 3000 87.9% chr2 + 136710654 136710714 61 browser details YourSeq 30 1068 1101 3000 96.9% chr2 + 130678671 130678705 35 browser details YourSeq 28 1056 1091 3000 88.9% chr1 - 125630796 125630831 36 browser details YourSeq 28 296 325 3000 96.7% chrX + 36002072 36002101 30 browser details YourSeq 28 1048 1094 3000 90.0% chr5 + 118966029 118966074 46 browser details YourSeq 26 313 345 3000 89.3% chr11 - 116990161 116990192 32 browser details YourSeq 26 91 129 3000 89.3% chrX + 21899100 21899137 38 browser details YourSeq 23 296 324 3000 89.7% chr11 + 3464155 3464183 29 browser details YourSeq 22 1066 1093 3000 89.3% chr7 - 38534834 38534861 28 browser details YourSeq 22 1009 1031 3000 100.0% chr3 - 142231469 142231496 28 browser details YourSeq 20 2014 2033 3000 100.0% chr12 - 57502296 57502315 20

Note: The 3000 bp section upstream of Exon 18 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% chr3 - 97744267 97747266 3000 browser details YourSeq 196 2785 2997 3000 96.3% chr3 - 97744142 97744386 245 browser details YourSeq 151 2838 3000 3000 96.4% chr3 - 97744171 97744333 163 browser details YourSeq 88 1927 2134 3000 74.4% chr8 + 104361169 104361356 188 browser details YourSeq 76 1923 2170 3000 70.3% chr17 + 34209787 34210003 217 browser details YourSeq 70 1985 2161 3000 70.6% chr11 + 61031543 61031691 149 browser details YourSeq 67 2047 2167 3000 77.7% chr6 - 86327199 86327319 121 browser details YourSeq 67 2044 2159 3000 79.2% chr10 - 95631145 95631261 117 browser details YourSeq 64 2052 2159 3000 79.7% chr1 - 177500756 177500863 108 browser details YourSeq 63 2045 2133 3000 85.4% chr2 - 32866426 32866514 89 browser details YourSeq 61 1908 2170 3000 74.4% chr10 - 119337304 119337505 202 browser details YourSeq 60 2028 2134 3000 77.5% chr17 + 47108283 47108388 106 browser details YourSeq 59 2029 2161 3000 79.3% chr5 - 20554811 20554932 122 browser details YourSeq 59 2043 2162 3000 79.3% chr17 - 27229269 27229416 148 browser details YourSeq 59 2001 2158 3000 92.8% chrX + 140057848 140058350 503 browser details YourSeq 59 2043 2133 3000 83.2% chr17 + 56231083 56231172 90 browser details YourSeq 58 2044 2134 3000 87.5% chr5 - 146310900 146310989 90 browser details YourSeq 58 2088 2166 3000 87.4% chr17 + 34620075 34673823 53749 browser details YourSeq 58 2045 2134 3000 82.3% chr15 + 100796909 100796998 90 browser details YourSeq 58 1981 2172 3000 87.2% chr10 + 126038245 126038750 506

Note: The 3000 bp section downstream of Exon 19 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Pde4dip phosphodiesterase 4D interacting protein (myomegalin) [ Mus musculus (house mouse) ] Gene ID: 83679, updated on 12-Aug-2019

Gene summary

Official Symbol Pde4dip provided by MGI Official Full Name phosphodiesterase 4D interacting protein (myomegalin) provided by MGI Primary source MGI:MGI:1891434 See related Ensembl:ENSMUSG00000038170 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 Usmg4; C87016; mKIAA0454; D3Bwg1078e; 4732458A06Rik; 9430063L05Rik; D130016K21Rik Expression Biased expression in heart adult (RPKM 75.3), mammary gland adult (RPKM 16.2) and 13 other tissues See more

Genomic context

Location: 3 F2.2; 3 42.28 cM See Pde4dip in Genome Data Viewer Exon count: 48

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (97689828..97888707, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (97493751..97692630, complement)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 9 transcripts

Gene: Pde4dip ENSMUSG00000038170

Description phosphodiesterase 4D interacting protein (myomegalin) [Source:MGI Symbol;Acc:MGI:1891434] Gene Synonyms 4732458A06Rik, 9430063L05Rik, D130016K21Rik, D3Bwg1078e, Usmg4 Location Chromosome 3: 97,689,824-97,888,707 reverse strand. GRCm38:CM000996.2 About this gene This gene has 9 transcripts (splice variants), 353 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pde4dip- ENSMUST00000090750.11 8376 2463aa ENSMUSP00000088254.5 Protein coding CCDS79985 G3X9L9 TSL:1 202 GENCODE basic APPRIS ALT2

Pde4dip- ENSMUST00000168438.8 8264 2412aa ENSMUSP00000131170.2 Protein coding CCDS79984 E9Q1A0 TSL:1 205 GENCODE basic APPRIS ALT2

Pde4dip- ENSMUST00000045243.14 6185 1118aa ENSMUSP00000040905.8 Protein coding CCDS38562 Q80YT7 TSL:1 201 GENCODE basic APPRIS P3

Pde4dip- ENSMUST00000107038.5 2826 834aa ENSMUSP00000102653.3 Protein coding - Q3UR03 CDS 3' 203 incomplete TSL:1

Pde4dip- ENSMUST00000200232.2 484 72aa ENSMUSP00000142941.1 Protein coding - A0A0G2JEX6 CDS 3' 209 incomplete TSL:2

Pde4dip- ENSMUST00000175751.7 4658 564aa ENSMUSP00000134832.1 Nonsense mediated - H3BJ38 TSL:1 206 decay

Pde4dip- ENSMUST00000163531.3 3845 No - Retained intron - - TSL:2 204 protein

Pde4dip- ENSMUST00000197791.1 536 No - lncRNA - - TSL:2 207 protein

Pde4dip- ENSMUST00000200063.1 414 No - lncRNA - - TSL:5 208 protein

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218.88 kb Forward strand 97.70Mb 97.75Mb 97.80Mb 97.85Mb Genes Gm31305-201 >lncRNA (Comprehensive set...

Gm31305-202 >lncRNA

Contigs AC130541.4 > AC153661.4 > AC131746.3 > Genes (Comprehensive set... < Pde4dip-205protein coding

< Pde4dip-208lncRNA < Pde4dip-201protein coding < Pde4dip-209protein coding < Gm15999-201lncRNA

< Pde4dip-202protein coding

< Mir7225-201miRNA < Pde4dip-206nonsense mediated decay < Pde4dip-204retained intron

< Pde4dip-207lncRNA < Pde4dip-203protein coding

Regulatory Build

97.70Mb 97.75Mb 97.80Mb 97.85Mb Reverse strand 218.88 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000090750

< Pde4dip-202protein coding

Reverse strand 198.84 kb

ENSMUSP00000088... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) SMART Olduvai domain Pfam Centrosomin, N-terminal motif 1 Olduvai domain

PROSITE profiles Olduvai domain PANTHER PTHR46501

PTHR46501:SF2

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 400 800 1200 1600 2000 2463

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

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