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Mouse Prr12 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Prr12 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Prr12 (NCBI Reference Sequence: NM_175022 ; Ensembl: ENSMUSG00000046574 ) is located on Mouse 7. 14 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000057293). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Prr12 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-373N14 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 2 starts from about 1.43% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1465 bp, and the size of intron 2 for 3'-loxP site insertion: 851 bp. The size of effective cKO region: ~613 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 2 3 4 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Prr12 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(7113bp) | A(17.03% 1211) | C(34.04% 2421) | T(21.9% 1558) | G(27.04% 1923)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr7 - 45051581 45054580 3000 browser details YourSeq 88 1233 1677 3000 76.7% chr13 + 118499994 118500178 185 browser details YourSeq 52 1236 1675 3000 61.7% chr8 + 81598486 81598547 62 browser details YourSeq 48 1232 1482 3000 71.5% chr1 + 52580835 52581051 217 browser details YourSeq 44 1233 1542 3000 64.0% chr5 + 119788062 119788239 178 browser details YourSeq 39 1159 1259 3000 81.4% chr5 + 52453338 52453431 94 browser details YourSeq 39 1240 1462 3000 66.7% chr14 + 11395924 11396066 143 browser details YourSeq 35 1515 1666 3000 56.1% chr13 - 41284520 41284563 44 browser details YourSeq 34 1644 1687 3000 94.8% chr5 + 136671181 136671227 47 browser details YourSeq 33 1100 1178 3000 94.5% chr5 + 113349631 113350146 516 browser details YourSeq 32 1235 1673 3000 42.9% chr17 - 48330334 48330368 35 browser details YourSeq 32 1158 1251 3000 97.2% chr6 + 62164590 62164686 97 browser details YourSeq 31 1438 1483 3000 72.3% chr6 + 5732021 5732057 37 browser details YourSeq 30 1235 1677 3000 40.6% chr2 + 23170703 23170739 37 browser details YourSeq 29 1644 1678 3000 94.3% chr16 - 95588549 95588592 44 browser details YourSeq 28 1646 1677 3000 96.7% chr5 - 147000724 147000756 33 browser details YourSeq 25 1133 1160 3000 84.7% chr12 - 68545471 68545496 26 browser details YourSeq 23 1234 1258 3000 87.5% chr19 + 39861443 39861466 24

Note: The 3000 bp section upstream of Exon 2 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% chr7 - 45047968 45050967 3000 browser details YourSeq 33 350 440 3000 97.2% chr9 - 49787011 49787150 140 browser details YourSeq 32 329 444 3000 94.5% chr10 + 67049856 67049971 116 browser details YourSeq 30 413 444 3000 96.9% chr2 - 152137341 152137372 32 browser details YourSeq 28 394 437 3000 81.9% chr7 + 67386784 67386827 44 browser details YourSeq 28 2849 2882 3000 87.5% chr16 + 37384376 37384408 33 browser details YourSeq 27 418 444 3000 100.0% chr7 - 38072496 38072522 27 browser details YourSeq 27 420 446 3000 100.0% chr14 - 57532390 57532416 27 browser details YourSeq 27 419 445 3000 100.0% chr5 + 142430030 142430056 27 browser details YourSeq 26 419 444 3000 100.0% chr2 - 162914071 162914096 26 browser details YourSeq 26 419 444 3000 100.0% chr14 - 60725626 60725651 26 browser details YourSeq 26 418 443 3000 100.0% chr10 - 80552956 80552981 26 browser details YourSeq 25 412 444 3000 87.9% chr7 + 137696466 137696498 33 browser details YourSeq 25 420 444 3000 100.0% chr15 + 100070683 100070707 25 browser details YourSeq 25 2858 2883 3000 100.0% chr1 + 56852879 56852906 28 browser details YourSeq 20 2888 2909 3000 95.5% chr5 + 144497540 144497561 22

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Prr12 proline rich 12 [ Mus musculus (house mouse) ] Gene ID: 233210, updated on 12-Aug-2019

Gene summary

Official Symbol Prr12 provided by MGI Official Full Name proline rich 12 provided by MGI Primary source MGI:MGI:2679002 See related Ensembl:ENSMUSG00000046574 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 BC058674; 6720469B10 Expression Ubiquitous expression in thymus adult (RPKM 11.0), adrenal adult (RPKM 9.5) and 27 other tissues See more Orthologs human all

Genomic context

Location: 7; 7 B3 See Prr12 in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (45027701..45053334, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (52283077..52308251, complement)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 2 transcripts

Gene: Prr12 ENSMUSG00000046574

Description proline rich 12 [Source:MGI Symbol;Acc:MGI:2679002] Location Chromosome 7: 45,027,563-45,052,881 reverse strand. GRCm38:CM001000.2 About this gene This gene has 2 transcripts (splice variants), 228 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Prr12-201 ENSMUST00000057293.7 7023 2035aa ENSMUSP00000054702.6 Protein coding CCDS52241 E9PYL2 TSL:5 GENCODE basic APPRIS P1

Prr12-202 ENSMUST00000210410.1 1487 No protein - Retained intron - - TSL:1

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45.32 kb Forward strand

45.02Mb 45.03Mb 45.04Mb 45.05Mb 45.06Mb Rras-201 >protein coding Nosip-205 >retained intron (Comprehensive set...

Rras-202 >nonsense mediated decay Nosip-204 >retained intron

Rras-203 >protein coding Nosip-202 >protein coding

Nosip-201 >protein coding

Nosip-210 >protein coding

Nosip-206 >protein coding

Nosip-207 >protein coding

Contigs AC126256.4 >

Genes (Comprehensive set... < Prr12-201protein coding < Prrg2-204nonsense mediated decay

< Prr12-202retained intron < Prrg2-208protein coding

< Prrg2-201protein coding

< Prrg2-210retained intron

< Prrg2-211protein coding

< Prrg2-207protein coding

< Prrg2-209protein coding

< Prrg2-203lncRNA

< Prrg2-206protein coding

< Prrg2-205protein coding

< Prrg2-202protein coding

Regulatory Build

45.02Mb 45.03Mb 45.04Mb 45.05Mb 45.06Mb Reverse strand 45.32 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000057293

< Prr12-201protein coding

Reverse strand 25.32 kb

ENSMUSP00000054... MobiDB lite Low complexity (Seg) Pfam Domain of unknown function DUF4211 PANTHER PTHR14709:SF1

PTHR14709

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

Variant Legend

inframe insertion inframe deletion missense variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2035

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

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