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Mouse Ppp2r5c Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ppp2r5c conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ppp2r5c gene (NCBI Reference Sequence: NM_012023 ; Ensembl: ENSMUSG00000017843 ) is located on Mouse chromosome 12. 14 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 14 (Transcript: ENSMUST00000084985). Exon 3~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ppp2r5c gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-143L24 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 gene-trapped allele show partial neonatal lethality, hypoactivity, and abnormal ventricular septum formation associated with increased fetal cardiomyocyte apoptosis. Surviving homozygotes develop obesity and show an abnormal gait, decreased grip strength, and impaired balance.

Exon 3 starts from about 18.77% of the coding region. The knockout of Exon 3~5 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 21384 bp, and the size of intron 5 for 3'-loxP site insertion: 5160 bp. The size of effective cKO region: ~2327 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 3 4 5 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ppp2r5c 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(8827bp) | A(23.25% 2052) | C(25.48% 2249) | T(27.26% 2406) | G(24.02% 2120)

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% chr12 + 110540826 110543825 3000 browser details YourSeq 164 1989 2466 3000 95.1% chr9 - 73060108 73218591 158484 browser details YourSeq 152 1990 2466 3000 94.3% chr17 - 57009123 57155424 146302 browser details YourSeq 148 2015 2466 3000 94.2% chr10 + 62949868 62950628 761 browser details YourSeq 147 2012 2462 3000 96.3% chr14 + 67003031 67003662 632 browser details YourSeq 146 2307 2484 3000 92.4% chr6 - 113973376 113973559 184 browser details YourSeq 141 2321 2474 3000 96.2% chr9 - 105063014 105063174 161 browser details YourSeq 140 2321 2476 3000 95.5% chr4 + 48649658 48650193 536 browser details YourSeq 138 2323 2471 3000 96.7% chr17 - 83618988 83619143 156 browser details YourSeq 137 2320 2474 3000 94.9% chr8 - 86822856 86823396 541 browser details YourSeq 137 2317 2471 3000 96.7% chr7 - 114183128 114183296 169 browser details YourSeq 137 2323 2466 3000 98.0% chr12 - 7585506 7585656 151 browser details YourSeq 137 2321 2466 3000 97.3% chr11 - 80427072 80427224 153 browser details YourSeq 137 2321 2464 3000 98.0% chr9 + 58566218 58566368 151 browser details YourSeq 137 2321 2474 3000 94.9% chr13 + 117349327 117349487 161 browser details YourSeq 134 2325 2471 3000 96.0% chr16 - 13790948 13791101 154 browser details YourSeq 134 2321 2466 3000 96.6% chr2 + 181791058 181791213 156 browser details YourSeq 133 2321 2471 3000 94.8% chr14 - 19838424 19838582 159 browser details YourSeq 132 2321 2471 3000 94.1% chr17 - 84208148 84208305 158 browser details YourSeq 132 2323 2466 3000 96.6% chr11 - 104325797 104325949 153

Note: The 3000 bp section upstream of Exon 3 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% chr12 + 110546153 110549152 3000 browser details YourSeq 160 2688 2905 3000 91.8% chr4 + 99129407 99129629 223 browser details YourSeq 145 2634 2906 3000 91.5% chr17 - 32961736 32962151 416 browser details YourSeq 139 2743 2957 3000 86.1% chr15 + 86212705 86212896 192 browser details YourSeq 137 2735 2900 3000 90.3% chr8 - 46553200 46553358 159 browser details YourSeq 137 2688 2902 3000 91.1% chr12 - 80349395 80349733 339 browser details YourSeq 137 2746 2906 3000 94.9% chr12 + 103087878 103088106 229 browser details YourSeq 136 2740 2899 3000 91.5% chr11 - 100350459 100350613 155 browser details YourSeq 135 2745 2901 3000 93.7% chr11 - 106497658 106637139 139482 browser details YourSeq 135 2734 2907 3000 91.0% chr9 + 44172991 44173164 174 browser details YourSeq 135 2688 2903 3000 85.5% chr7 + 102142653 102142846 194 browser details YourSeq 135 2688 2900 3000 85.3% chr2 + 34801149 34801329 181 browser details YourSeq 135 2742 2914 3000 90.0% chr11 + 55385874 55386044 171 browser details YourSeq 134 2740 2901 3000 90.2% chr9 - 108543608 108543763 156 browser details YourSeq 134 2688 2901 3000 90.4% chr7 - 30545088 30545340 253 browser details YourSeq 134 2740 2903 3000 89.3% chr5 - 111319376 111319534 159 browser details YourSeq 134 2740 2908 3000 93.0% chr11 + 45704885 45705054 170 browser details YourSeq 134 2679 2901 3000 93.0% chr10 + 99192104 99192483 380 browser details YourSeq 133 2743 2901 3000 90.2% chr19 - 5886319 5886472 154 browser details YourSeq 133 2742 2900 3000 90.8% chr18 - 34560013 34560166 154

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ppp2r5c protein phosphatase 2, regulatory subunit B', gamma [ Mus musculus (house mouse) ] Gene ID: 26931, updated on 24-Oct-2019

Gene summary

Official Symbol Ppp2r5c provided by MGI Official Full Name protein phosphatase 2, regulatory subunit B', gamma provided by MGI Primary source MGI:MGI:1349475 See related Ensembl:ENSMUSG00000017843 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 C85228; AI060890; AW545884; D12Bwg0916e; 2610043M05Rik; 2700063L20Rik Expression Ubiquitous expression in testis adult (RPKM 41.8), cortex adult (RPKM 18.2) and 27 other tissues See more Orthologs human all

Genomic context

Location: 12 F1; 12 60.56 cM See Ppp2r5c in Genome Data Viewer

Exon count: 18

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (110447144..110583075)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (111685389..111821271)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 7 transcripts

Gene: Ppp2r5c ENSMUSG00000017843

Description protein phosphatase 2, regulatory subunit B', gamma [Source:MGI Symbol;Acc:MGI:1349475] Gene Synonyms 2610043M05Rik, 2700063L20Rik, B56/PP2A gamma, Band 8A, D12Bwg0916e Location Chromosome 12: 110,447,120-110,583,062 forward strand. GRCm38:CM001005.2 About this gene This gene has 7 transcripts (splice variants), 220 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 19 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ppp2r5c- ENSMUST00000109832.2 4108 485aa ENSMUSP00000105458.1 Protein coding CCDS36558 Q60996 TSL:2 202 GENCODE basic APPRIS ALT1

Ppp2r5c- ENSMUST00000084985.10 1847 524aa ENSMUSP00000082053.3 Protein coding CCDS36557 Q60996 TSL:2 201 GENCODE basic APPRIS P4

Ppp2r5c- ENSMUST00000221715.1 2367 452aa ENSMUSP00000152865.1 Protein coding - Q60996 TSL:1 205 GENCODE basic

Ppp2r5c- ENSMUST00000221074.1 1971 496aa ENSMUSP00000152282.1 Protein coding - Q60996 TSL:5 204 GENCODE basic

Ppp2r5c- ENSMUST00000222276.1 1802 49aa ENSMUSP00000152369.1 Protein coding - A0A1Y7VJC8 TSL:2 206 GENCODE basic

Ppp2r5c- ENSMUST00000220509.1 956 283aa ENSMUSP00000152095.1 Protein coding - A0A1Y7VIR0 CDS 3' incomplete 203 TSL:5

Ppp2r5c- ENSMUST00000223168.1 2790 No - Retained intron - - TSL:2 207 protein

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155.94 kb Forward strand 110.44Mb 110.46Mb 110.48Mb 110.50Mb 110.52Mb 110.54Mb 110.56Mb 110.58Mb Genes (Comprehensive set... Ppp2r5c-206 >protein coding Ppp2r5c-205 >protein coding

Ppp2r5c-203 >protein coding B930059L03Rik-201 >lncRNA

Ppp2r5c-207 >retained intron

Ppp2r5c-202 >protein coding

Ppp2r5c-204 >protein coding

Ppp2r5c-201 >protein coding

Contigs AL591582.32 > AC152827.4 > Genes < Gm19605-202lncRNA (Comprehensive set...

< Gm19605-203lncRNA

< Gm19605-205lncRNA

< Gm19605-201lncRNA

< Gm19605-204retained intron

< Gm19605-207lncRNA

< Gm19605-206lncRNA

< Gm19605-208retained intron

Regulatory Build

110.44Mb 110.46Mb 110.48Mb 110.50Mb 110.52Mb 110.54Mb 110.56Mb 110.58Mb Reverse strand 155.94 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

RNA gene processed transcript

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Transcript: ENSMUST00000084985

94.95 kb Forward strand

Ppp2r5c-201 >protein coding

ENSMUSP00000082... MobiDB lite Low complexity (Seg) Superfamily Armadillo-type fold

Pfam Protein phosphatase 2A, regulatory B subunit, B56

PIRSF Protein phosphatase 2A, regulatory B subunit, B56

PANTHER Protein phosphatase 2A, regulatory B subunit, B56

PTHR10257:SF48 Gene3D Armadillo-like helical

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

Variant Legend

splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 524

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

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