https://www.alphaknockout.com

Mouse Ppp2r2a Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ppp2r2a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ppp2r2a gene (NCBI Reference Sequence: NM_028032 ; Ensembl: ENSMUSG00000022052 ) is located on Mouse chromosome 14. 10 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000089230). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ppp2r2a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-62C15 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:

Exon 4 starts from about 13.5% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 9844 bp, and the size of intron 4 for 3'-loxP site insertion: 4224 bp. The size of effective cKO region: ~666 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 4 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ppp2r2a Homology arm cKO region loxP site

Page 2 of 7 https://www.alphaknockout.com

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(7166bp) | A(26.46% 1896) | C(18.11% 1298) | T(34.13% 2446) | G(21.3% 1526)

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.

Page 3 of 7 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr14 - 67029281 67032280 3000 browser details YourSeq 139 1230 1396 3000 94.5% chr4 - 106844723 106845094 372 browser details YourSeq 139 1217 1399 3000 89.8% chr16 - 95518244 95518460 217 browser details YourSeq 134 1227 1380 3000 94.8% chr12 - 111472212 111472397 186 browser details YourSeq 133 1104 1380 3000 92.4% chr2 - 72968172 72968602 431 browser details YourSeq 132 1225 1392 3000 91.9% chr9 - 22466604 22466802 199 browser details YourSeq 126 1228 1377 3000 93.9% chr10 - 12885129 12885307 179 browser details YourSeq 125 1222 1380 3000 91.4% chr12 + 22018235 22018418 184 browser details YourSeq 125 1227 1413 3000 91.9% chr10 + 111256920 111257106 187 browser details YourSeq 124 1225 1399 3000 88.8% chr13 - 119441680 119441932 253 browser details YourSeq 123 1230 1380 3000 94.3% chr2 - 180649581 180649752 172 browser details YourSeq 123 1228 1380 3000 94.9% chr11 - 90688925 90689106 182 browser details YourSeq 122 1218 1380 3000 90.7% chr9 - 58235523 58235706 184 browser details YourSeq 122 1217 1380 3000 90.7% chr5 + 79970961 79971155 195 browser details YourSeq 121 1217 1380 3000 89.1% chr13 - 115883347 115883548 202 browser details YourSeq 121 1218 1380 3000 91.1% chr4 + 143236345 143236521 177 browser details YourSeq 118 1227 1360 3000 94.1% chr13 - 24403951 24404084 134 browser details YourSeq 117 1227 1380 3000 93.4% chr15 + 83096695 83096874 180 browser details YourSeq 116 1242 1380 3000 92.1% chr9 - 101917794 101917951 158 browser details YourSeq 116 1227 1358 3000 94.7% chr17 - 74459322 74459488 167

Note: The 3000 bp section upstream of Exon 4 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% chr14 - 67025615 67028614 3000 browser details YourSeq 176 1604 1795 3000 96.4% chr14 - 73229455 73229659 205 browser details YourSeq 175 1583 1776 3000 96.4% chr8 + 111647670 111647874 205 browser details YourSeq 173 1589 1783 3000 94.9% chr11 + 95295621 95295830 210 browser details YourSeq 172 1590 1790 3000 93.9% chr16 + 13803452 13803658 207 browser details YourSeq 171 1590 1793 3000 93.7% chr2 + 79721943 79722144 202 browser details YourSeq 169 1594 1776 3000 96.2% chr5 - 137479017 137479199 183 browser details YourSeq 168 1594 1775 3000 96.2% chr19 - 7007499 7007680 182 browser details YourSeq 168 1596 1775 3000 97.3% chr16 + 38382898 38383503 606 browser details YourSeq 168 1590 1776 3000 92.9% chr10 + 115895229 115895409 181 browser details YourSeq 167 1590 1775 3000 95.7% chr9 - 72126619 72126990 372 browser details YourSeq 167 1594 1784 3000 93.8% chr11 + 88858046 88858236 191 browser details YourSeq 166 1602 1789 3000 94.2% chr9 + 100632841 100633028 188 browser details YourSeq 166 1590 1775 3000 95.2% chr7 + 28625928 28626127 200 browser details YourSeq 166 1597 1775 3000 96.7% chr12 + 70974154 70974333 180 browser details YourSeq 165 1604 1783 3000 96.7% chr4 + 40838574 40838754 181 browser details YourSeq 164 1589 1775 3000 91.7% chr2 + 157226713 157226893 181 browser details YourSeq 164 1583 1774 3000 93.7% chr10 + 37700003 37700198 196 browser details YourSeq 163 1594 1783 3000 93.7% chrX - 150227131 150227320 190 browser details YourSeq 163 1589 1775 3000 91.9% chr8 + 22412875 22413058 184

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 7 https://www.alphaknockout.com

Gene and protein information: Ppp2r2a protein phosphatase 2, regulatory subunit B, alpha [ Mus musculus (house mouse) ] Gene ID: 71978, updated on 24-Oct-2019

Gene summary

Official Symbol Ppp2r2a provided by MGI Official Full Name protein phosphatase 2, regulatory subunit B, alpha provided by MGI Primary source MGI:MGI:1919228 See related Ensembl:ENSMUSG00000022052 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 mKIAA1541; 2410004D02Rik Expression Ubiquitous expression in adrenal adult (RPKM 26.6), CNS E11.5 (RPKM 11.2) and 27 other tissues See more Orthologs human all

Genomic context

Location: 14; 14 D1 See Ppp2r2a in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (67014056..67072471, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (67634508..67691266, complement)

Chromosome 14 - NC_000080.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 5 transcripts

Gene: Ppp2r2a ENSMUSG00000022052

Description protein phosphatase 2, regulatory subunit B, alpha [Source:MGI Symbol;Acc:MGI:1919228] Gene Synonyms 2410004D02Rik Location Chromosome 14: 67,014,056-67,072,444 reverse strand. GRCm38:CM001007.2 About this gene This gene has 5 transcripts (splice variants), 146 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ppp2r2a- ENSMUST00000089230.6 4010 447aa ENSMUSP00000086640.5 Protein coding CCDS56967 Q3TT94 TSL:1 201 Q6P1F6 GENCODE basic APPRIS P1

Ppp2r2a- ENSMUST00000225380.1 1988 367aa ENSMUSP00000153191.1 Protein coding - Q9CWU3 GENCODE 204 basic

Ppp2r2a- ENSMUST00000225251.1 2192 50aa ENSMUSP00000153452.1 Nonsense mediated - A0A286YDJ9 - 203 decay

Ppp2r2a- ENSMUST00000223630.1 1845 No - Retained intron - - - 202 protein

Ppp2r2a- ENSMUST00000225469.1 770 No - lncRNA - - - 205 protein

78.39 kb Forward strand

Genes Gm27647-201 >miRNA (Comprehensive set...

Contigs AC165148.2 > < CT030233.6

Genes (Comprehensive set... < Bnip3l-201protein codin

< Bnip3l-202protein coding< Ppp2r2a-203nonsense mediated decay

< Ppp2r2a-201protein coding

< Ppp2r2a-202retained intron

< Ppp2r2a-205lncRNA

Regulatory Build

Reverse strand 78.39 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000089230

< Ppp2r2a-201protein coding

Reverse strand 58.39 kb

ENSMUSP00000086... Low complexity (Seg) Superfamily WD40-repeat-containing domain superfamily

SMART WD40 repeat Prints Protein phosphatase 2A regulatory subunit PR55 PROSITE patterns Protein phosphatase 2A regulatory subunit PR55, conserved site

Protein phosphatase 2A regulatory subunit PR55, conserved site PIRSF Protein phosphatase 2A regulatory subunit PR55

PANTHER Protein phosphatase 2A regulatory subunit PR55

PTHR11871:SF2 Gene3D WD40/YVTN repeat-like-containing domain superfamily

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

Variant Legend splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 447

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

Page 7 of 7