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Mouse Ppp1r12a Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ppp1r12a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ppp1r12a gene (NCBI Reference Sequence: NM_027892 ; Ensembl: ENSMUSG00000019907 ) is located on Mouse chromosome 10. 26 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 25 (Transcript: ENSMUST00000070663). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ppp1r12a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-413N6 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 die before E7.5. Mice homozygous for a floxed allele activated in smooth muscle exhibit altered intestinal smooth muscle contractility.

Exon 3 starts from about 12.25% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 15424 bp, and the size of intron 3 for 3'-loxP site insertion: 15930 bp. The size of effective cKO region: ~619 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 3 26 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ppp1r12a 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(7119bp) | A(34.6% 2463) | C(18.12% 1290) | T(28.35% 2018) | G(18.94% 1348)

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% chr10 + 108211139 108214138 3000 browser details YourSeq 161 444 2337 3000 96.1% chr12 + 83501340 83621858 120519 browser details YourSeq 134 442 586 3000 97.3% chr2 - 38561538 38561704 167 browser details YourSeq 131 184 578 3000 83.1% chr18 + 48377653 48377836 184 browser details YourSeq 131 444 585 3000 97.2% chr1 + 150030359 150030513 155 browser details YourSeq 130 444 588 3000 95.9% chr12 - 78899461 78899617 157 browser details YourSeq 130 449 585 3000 97.9% chr15 + 101117621 101117766 146 browser details YourSeq 129 443 589 3000 94.6% chr9 - 59742026 59742182 157 browser details YourSeq 128 445 585 3000 96.5% chr11 - 102115544 102115692 149 browser details YourSeq 128 444 585 3000 95.8% chr11 - 46053926 46054077 152 browser details YourSeq 128 434 582 3000 93.9% chr17 + 15844753 15844912 160 browser details YourSeq 127 442 585 3000 96.4% chr7 + 25029889 25030036 148 browser details YourSeq 127 442 583 3000 95.8% chr2 + 86040908 86041061 154 browser details YourSeq 125 442 586 3000 94.5% chr15 + 57436508 57436661 154 browser details YourSeq 124 444 585 3000 94.4% chr5 + 129672583 129672734 152 browser details YourSeq 124 445 585 3000 95.0% chr13 + 89151259 89151407 149 browser details YourSeq 122 442 571 3000 97.7% chr15 + 90901658 90901797 140 browser details YourSeq 122 455 849 3000 83.9% chr11 + 121538296 121538434 139 browser details YourSeq 121 444 572 3000 97.7% chr7 + 80016273 80016411 139 browser details YourSeq 120 445 578 3000 96.3% chr14 - 19997579 19997721 143

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% chr10 + 108214758 108217757 3000 browser details YourSeq 1727 1163 3000 3000 97.2% chr15 - 36134577 36136417 1841 browser details YourSeq 1718 1163 3000 3000 97.0% chr6 - 22750957 22752797 1841 browser details YourSeq 1715 1163 3000 3000 96.9% chr9 - 123618481 123620321 1841 browser details YourSeq 1712 1163 3000 3000 96.8% chr18 + 41420200 41422040 1841 browser details YourSeq 1707 1163 3000 3000 96.7% chr6 - 106363599 106365438 1840 browser details YourSeq 1700 1163 3000 3000 96.5% chr2 - 76278494 76280335 1842 browser details YourSeq 1699 1163 3000 3000 96.4% chr13 - 42128351 42130189 1839 browser details YourSeq 1698 1163 2999 3000 96.4% chr2 - 85369303 85371141 1839 browser details YourSeq 1698 1163 3000 3000 96.4% chr4 + 85189209 85191046 1838 browser details YourSeq 1697 1163 3000 3000 96.4% chr5 - 110067274 110069115 1842 browser details YourSeq 1694 1163 3000 3000 96.3% chr10 - 58723950 58780900 56951 browser details YourSeq 1692 1163 3000 3000 96.3% chr13 + 4923982 4925823 1842 browser details YourSeq 1691 1163 3000 3000 96.4% chr6 - 57671780 57673619 1840 browser details YourSeq 1691 1163 3000 3000 96.3% chr16 + 76920205 76922048 1844 browser details YourSeq 1689 1173 3000 3000 96.4% chr10 - 104620548 104622376 1829 browser details YourSeq 1689 1163 3000 3000 96.2% chr11 + 17099002 17100842 1841 browser details YourSeq 1688 1163 3000 3000 96.1% chr6 - 126416310 126418148 1839 browser details YourSeq 1688 1163 3000 3000 96.1% chr11 - 30072080 30073917 1838 browser details YourSeq 1682 1169 3000 3000 96.4% chrX - 97225289 97227124 1836

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: Ppp1r12a protein phosphatase 1, regulatory subunit 12A [ Mus musculus (house mouse) ] Gene ID: 17931, updated on 24-Oct-2019

Gene summary

Official Symbol Ppp1r12a provided by MGI Official Full Name protein phosphatase 1, regulatory subunit 12A provided by MGI Primary source MGI:MGI:1309528 See related Ensembl:ENSMUSG00000019907 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 Mypt1; AA792106; AV099298; D10Ertd625e; 1200015F06Rik; 5730577I22Rik Expression Biased expression in bladder adult (RPKM 144.2), CNS E11.5 (RPKM 14.0) and 11 other tissues See more Orthologs human all

Genomic context

Location: 10 D1; 10 56.33 cM See Ppp1r12a in Genome Data Viewer

Exon count: 26

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (108162092..108279985)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (107599456..107714631)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 8 transcripts

Gene: Ppp1r12a ENSMUSG00000019907

Description protein phosphatase 1, regulatory subunit 12A [Source:MGI Symbol;Acc:MGI:1309528] Gene Synonyms 1200015F06Rik, 5730577I22Rik, D10Ertd625e, Mypt1 Location Chromosome 10: 108,162,193-108,284,475 forward strand. GRCm38:CM001003.2 About this gene This gene has 8 transcripts (splice variants), 200 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ppp1r12a- ENSMUST00000070663.5 3403 1004aa ENSMUSP00000069257.5 Protein coding CCDS36052 Q9DBR7 TSL:1 201 GENCODE basic

Ppp1r12a- ENSMUST00000219759.1 4212 506aa ENSMUSP00000151478.1 Protein coding - A0A1W2P750 CDS 5' 208 incomplete TSL:1

Ppp1r12a- ENSMUST00000219263.1 3355 1029aa ENSMUSP00000151842.1 Protein coding - Q9DBR7 TSL:5 205 GENCODE basic APPRIS P1

Ppp1r12a- ENSMUST00000218161.1 5066 No - Retained - - TSL:2 202 protein intron

Ppp1r12a- ENSMUST00000219068.1 590 No - Retained - - TSL:3 204 protein intron

Ppp1r12a- ENSMUST00000219653.1 563 No - Retained - - TSL:3 207 protein intron

Ppp1r12a- ENSMUST00000219559.1 541 No - lncRNA - - TSL:3 206 protein

Ppp1r12a- ENSMUST00000218773.1 386 No - lncRNA - - TSL:2 203 protein

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142.28 kb Forward strand 108.16Mb 108.18Mb 108.20Mb 108.22Mb 108.24Mb 108.26Mb 108.28Mb Genes (Comprehensive set... Ppp1r12a-205 >protein coding

Ppp1r12a-201 >protein coding

Ppp1r12a-208 >protein coding

Ppp1r12a-203 >lncRNA Ppp1r12a-206 >lncRNA

Ppp1r12a-204 >retained intron

Ppp1r12a-202 >retained intron

Ppp1r12a-207 >retained intron

Contigs AC139335.3 > Regulatory Build

108.16Mb 108.18Mb 108.20Mb 108.22Mb 108.24Mb 108.26Mb 108.28Mb Reverse strand 142.28 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

processed transcript RNA gene

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Transcript: ENSMUST00000070663

115.18 kb Forward strand

Ppp1r12a-201 >protein coding

ENSMUSP00000069... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Ankyrin repeat-containing domain superfamily SMART Ankyrin repeat Prints Ankyrin repeat Pfam Ankyrin repeat-containing domain cGMP-dependent protein kinase, interacting domain PROSITE profiles Ankyrin repeat-containing domain

Ankyrin repeat PIRSF Protein phosphatase 1 regulatory subunit 12A/B/C PANTHER PTHR24179

PTHR24179:SF20 Gene3D Ankyrin repeat-containing domain superfamily

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1004

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

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