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Mouse Ppp1r9a Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ppp1r9a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ppp1r9a gene (NCBI Reference Sequence: NM_181595 ; Ensembl: ENSMUSG00000032827 ) is located on Mouse chromosome 6. 16 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000035813). 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 Ppp1r9a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-2F8 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 knock-out allele exhibit defects in dopamine-mediated neuromodulation, deficient long-term potentiation at corticostriatal synapses, increased spontaneous excitatory post-synaptic current frequency, and enhanced locomotor activationin response to cocaine treatment.

Exon 3 starts from about 42.59% 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: 139091 bp, and the size of intron 3 for 3'-loxP site insertion: 11389 bp. The size of effective cKO region: ~633 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 16 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ppp1r9a 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7133bp) | A(28.63% 2042) | C(18.72% 1335) | T(30.72% 2191) | G(21.94% 1565)

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 + 5042687 5045686 3000 browser details YourSeq 165 165 364 3000 92.8% chr13 + 94810081 94810286 206 browser details YourSeq 165 148 364 3000 88.1% chr11 + 66794255 66794467 213 browser details YourSeq 161 167 364 3000 93.1% chr10 - 22099067 22099267 201 browser details YourSeq 161 170 365 3000 92.2% chrX + 168050299 168050516 218 browser details YourSeq 161 173 381 3000 93.1% chr16 + 16943055 16943422 368 browser details YourSeq 160 142 364 3000 91.3% chr10 - 32231708 32232077 370 browser details YourSeq 157 170 359 3000 93.5% chr2 + 48176390 48176587 198 browser details YourSeq 157 167 357 3000 91.9% chr1 + 44631705 44631894 190 browser details YourSeq 156 168 364 3000 92.0% chr2 - 13717342 13717540 199 browser details YourSeq 156 155 359 3000 87.2% chr4 + 82261838 82262035 198 browser details YourSeq 156 173 364 3000 92.4% chr4 + 63818488 63818680 193 browser details YourSeq 155 173 364 3000 91.2% chr11 + 82506596 82506785 190 browser details YourSeq 154 166 358 3000 92.4% chr14 - 103006466 103006662 197 browser details YourSeq 154 173 364 3000 92.9% chr2 + 99421987 99422178 192 browser details YourSeq 154 173 358 3000 93.8% chr13 + 112170205 112170390 186 browser details YourSeq 153 173 364 3000 91.4% chrX + 153654951 153655148 198 browser details YourSeq 153 169 359 3000 92.6% chr18 + 10374346 10374534 189 browser details YourSeq 152 173 355 3000 90.5% chr3 - 88759135 88759314 180 browser details YourSeq 152 172 359 3000 91.4% chr1 + 65018860 65019051 192

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% chr6 + 5046320 5049319 3000 browser details YourSeq 114 69 475 3000 92.6% chr16 - 22143506 22143912 407 browser details YourSeq 99 62 283 3000 89.1% chr5 - 31071072 31071582 511 browser details YourSeq 94 78 492 3000 91.4% chr16 + 97192041 97192625 585 browser details YourSeq 93 78 499 3000 77.6% chr12 - 8353847 8354121 275 browser details YourSeq 93 62 196 3000 90.6% chr11 + 53220176 53220348 173 browser details YourSeq 90 78 196 3000 91.8% chr2 - 161114620 161114759 140 browser details YourSeq 89 62 166 3000 93.3% chr17 - 68215171 68215290 120 browser details YourSeq 87 62 166 3000 92.4% chr17 - 45719087 45719206 120 browser details YourSeq 87 88 212 3000 87.3% chr13 - 100845801 100845945 145 browser details YourSeq 85 62 165 3000 92.4% chr12 + 21532218 21532336 119 browser details YourSeq 84 69 190 3000 90.5% chr12 + 54123611 54123769 159 browser details YourSeq 83 62 166 3000 90.4% chrX - 100918181 100918300 120 browser details YourSeq 81 56 166 3000 88.8% chr7 + 143543042 143543166 125 browser details YourSeq 81 83 283 3000 82.2% chr7 + 126304480 126304655 176 browser details YourSeq 81 69 166 3000 92.7% chr16 + 91719470 91719580 111 browser details YourSeq 81 62 166 3000 89.4% chr15 + 36109707 36109825 119 browser details YourSeq 80 62 190 3000 89.4% chr2 - 147730723 147730869 147 browser details YourSeq 80 62 165 3000 89.5% chr10 - 100119956 100120067 112 browser details YourSeq 80 69 166 3000 91.9% chr13 + 59277801 59277905 105

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: Ppp1r9a protein phosphatase 1, regulatory subunit 9A [ Mus musculus (house mouse) ] Gene ID: 243725, updated on 24-Oct-2019

Gene summary

Official Symbol Ppp1r9a provided by MGI Official Full Name protein phosphatase 1, regulatory subunit 9A provided by MGI Primary source MGI:MGI:2442401 See related Ensembl:ENSMUSG00000032827 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 NRB; BB181831; 5330407E15; neurabin-I; 2810430P21Rik; 4930518N04Rik; A230094E16Rik Expression Broad expression in cortex adult (RPKM 12.5), frontal lobe adult (RPKM 11.0) and 22 other tissues See more Orthologs human all

Genomic context

Location: 6 A1; 6 1.86 cM See Ppp1r9a in Genome Data Viewer

Exon count: 25

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (4902872..5165661)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (4853320..5115661)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 12 transcripts

Gene: Ppp1r9a ENSMUSG00000032827

Description protein phosphatase 1, regulatory subunit 9A [Source:MGI Symbol;Acc:MGI:2442401] Gene Synonyms 2810430P21Rik, 4930518N04Rik, A230094E16Rik, NRB, Neurabin I, neurabin-I Location Chromosome 6: 4,902,917-5,165,661 forward strand. GRCm38:CM000999.2 About this gene This gene has 12 transcripts (splice variants), 210 orthologues, 2 paralogues, 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

Ppp1r9a- ENSMUST00000035813.8 9547 1095aa ENSMUSP00000046906.2 Protein coding CCDS19897 Q7TN74 TSL:1 201 GENCODE basic APPRIS P2

Ppp1r9a- ENSMUST00000177456.7 3975 1292aa ENSMUSP00000134943.1 Protein coding - H3BJD6 TSL:5 212 GENCODE basic APPRIS ALT2

Ppp1r9a- ENSMUST00000175962.1 3769 533aa ENSMUSP00000135360.1 Protein coding - H3BKE7 TSL:1 206 GENCODE basic

Ppp1r9a- ENSMUST00000175889.7 3462 1042aa ENSMUSP00000135629.1 Protein coding - H3BL28 CDS 3' 205 incomplete TSL:1

Ppp1r9a- ENSMUST00000168998.8 2966 642aa ENSMUSP00000126643.2 Protein coding - Q3UXW4 TSL:1 204 GENCODE basic

Ppp1r9a- ENSMUST00000177338.1 1680 447aa ENSMUSP00000135634.1 Protein coding - Q8BMP0 CDS 3' 211 incomplete TSL:1

Ppp1r9a- ENSMUST00000176263.7 4810 977aa ENSMUSP00000134937.1 Nonsense mediated - H3BJD0 TSL:5 208 decay

Ppp1r9a- ENSMUST00000177153.7 3834 955aa ENSMUSP00000135485.1 Nonsense mediated - H3BKQ7 TSL:5 210 decay

Ppp1r9a- ENSMUST00000176729.7 3046 232aa ENSMUSP00000134909.1 Nonsense mediated - H3BJA6 CDS 5' 209 decay incomplete TSL:1

Ppp1r9a- ENSMUST00000164110.8 9395 No - Retained intron - - TSL:1 203 protein

Ppp1r9a- ENSMUST00000065842.6 885 No - Retained intron - - TSL:1 202 protein

Ppp1r9a- ENSMUST00000176136.1 357 No - lncRNA - - TSL:2 207 protein

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282.75 kb Forward strand

4.9Mb 5.0Mb 5.1Mb Genes (Comprehensive set... Ppp1r9a-205 >protein coding

Ppp1r9a-204 >protein coding Ppp1r9a-207 >lncRNA

Ppp1r9a-208 >nonsense mediated decay

Ppp1r9a-201 >protein coding

Ppp1r9a-206 >protein coding Ppp1r9a-202 >retained intron

Ppp1r9a-203 >retained intron

Ppp1r9a-211 >protein coGdimng26814-201 >TEC

Ppp1r9a-212 >protein coding

Ppp1r9a-210 >nonsense mediated decay

Ppp1r9a-209 >nonsense mediated decay

Gm26736-201 >TEC

Gm26556-201 >TEC

Contigs AC068500.4 > AC074225.4 > Genes < Gmfg-ps-201processed pseudogene < Pon1-201protein coding (Comprehensive set...

< Pon1-204protein coding

< Pon1-203protein coding

Regulatory Build

4.9Mb 5.0Mb 5.1Mb Reverse strand 282.75 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

processed transcript pseudogene RNA gene

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Transcript: ENSMUST00000035813

262.34 kb Forward strand

Ppp1r9a-201 >protein coding

ENSMUSP00000046... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily PDZ superfamily Sterile alpha motif/pointed domain superfamily

SMART PDZ domain Sterile alpha motif domain

Pfam PDZ domain Sterile alpha motif domain

Neurabin-1/2, PDZ domain PROSITE profiles PDZ domain Sterile alpha motif domain

PANTHER Neurabin-1

PTHR16154 Gene3D 2.30.42.10 Sterile alpha motif/pointed domain superfamily

CDD cd00992 cd09512

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

Variant Legend

inframe insertion missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1095

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

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