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Mouse Parp12 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Parp12 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Parp12 (NCBI Reference Sequence: NM_172893 ; Ensembl: ENSMUSG00000038507 ) is located on Mouse 6. 12 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 12 (Transcript: ENSMUST00000038398). 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 Parp12 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-412O23 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 16.6% 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: 3505 bp, and the size of intron 2 for 3'-loxP site insertion: 2354 bp. The size of effective cKO region: ~636 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 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Parp12 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(7136bp) | A(23.82% 1700) | C(20.17% 1439) | T(30.97% 2210) | G(25.04% 1787)

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 - 39114552 39117551 3000 browser details YourSeq 215 1978 2514 3000 86.3% chr11 - 4894620 4894919 300 browser details YourSeq 211 1979 2517 3000 83.2% chr11 + 4914956 4915286 331 browser details YourSeq 194 1978 2498 3000 88.3% chr18 - 66423996 66424678 683 browser details YourSeq 193 1978 2475 3000 83.8% chr1 - 152951213 152951470 258 browser details YourSeq 189 1978 2517 3000 93.2% chr14 + 25953875 26233200 279326 browser details YourSeq 186 1978 2510 3000 91.2% chr14 - 27358811 27359452 642 browser details YourSeq 184 1978 2517 3000 82.8% chr9 + 57618203 57618614 412 browser details YourSeq 180 1977 2331 3000 85.7% chr12 - 110671623 110671951 329 browser details YourSeq 180 1978 2517 3000 86.4% chr5 + 150643503 150644019 517 browser details YourSeq 177 1980 2515 3000 91.6% chr12 - 65192261 65192805 545 browser details YourSeq 175 1978 2398 3000 90.4% chr13 - 55145093 55364641 219549 browser details YourSeq 174 1978 2450 3000 91.5% chr9 - 108659443 108660071 629 browser details YourSeq 173 1978 2174 3000 94.9% chr4 - 32505607 32771780 266174 browser details YourSeq 166 1978 2500 3000 87.4% chr6 + 39593856 39594312 457 browser details YourSeq 166 1980 2193 3000 89.2% chr11 + 71120569 71120767 199 browser details YourSeq 164 1978 2474 3000 91.5% chr8 + 70705713 70706371 659 browser details YourSeq 162 1963 2161 3000 90.8% chr9 - 72211040 72211228 189 browser details YourSeq 162 1980 2517 3000 91.4% chr11 + 96068861 96069430 570 browser details YourSeq 161 1979 2163 3000 94.0% chr4 - 150622476 150622663 188

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% chr6 - 39110916 39113915 3000 browser details YourSeq 48 1108 1179 3000 91.6% chr3 + 31030106 31030182 77 browser details YourSeq 45 390 734 3000 94.0% chr7 - 87343587 87343966 380 browser details YourSeq 44 1695 1781 3000 92.4% chr10 - 81621645 81621731 87 browser details YourSeq 34 1691 1779 3000 92.7% chr17 - 4953637 4953728 92 browser details YourSeq 31 1765 1795 3000 100.0% chr11 - 98532416 98532446 31 browser details YourSeq 30 1765 1794 3000 100.0% chr6 - 88739072 88739101 30 browser details YourSeq 30 1765 1796 3000 96.9% chr10 - 43975495 43975526 32 browser details YourSeq 30 1696 1787 3000 94.2% chr7 + 123116860 123116951 92 browser details YourSeq 30 1755 1787 3000 97.0% chr1 + 151304243 151304287 45 browser details YourSeq 29 1764 1792 3000 100.0% chr8 - 47298834 47298862 29 browser details YourSeq 29 1767 1795 3000 100.0% chr2 - 26290140 26290168 29 browser details YourSeq 29 1765 1795 3000 96.8% chr10 - 45368055 45368085 31 browser details YourSeq 29 1766 1796 3000 96.8% chrX + 50951116 50951146 31 browser details YourSeq 29 1765 1793 3000 100.0% chr4 + 128090695 128090723 29 browser details YourSeq 28 400 441 3000 91.0% chr2 + 45198270 45198311 42 browser details YourSeq 26 1768 1793 3000 100.0% chr11 + 88077715 88077740 26 browser details YourSeq 26 1765 1790 3000 100.0% chr1 + 179811206 179811231 26 browser details YourSeq 24 1765 1792 3000 92.9% chr6 - 119872490 119872517 28 browser details YourSeq 24 1765 1794 3000 90.0% chr3 - 21791745 21791774 30

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 protein information: Parp12 poly (ADP-ribose) polymerase family, member 12 [ Mus musculus (house mouse) ] Gene ID: 243771, updated on 12-Aug-2019

Gene summary

Official Symbol Parp12 provided by MGI Official Full Name poly (ADP-ribose) polymerase family, member 12 provided by MGI Primary source MGI:MGI:2143990 See related Ensembl:ENSMUSG00000038507 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 ARTD12; PARP-12; Zc3hdc1; AA409132; AA536654; 9930021O16 Expression Ubiquitous expression in small intestine adult (RPKM 28.4), colon adult (RPKM 27.7) and 26 other tissues See more Orthologs all

Genomic context

Location: 6; 6 B1 See Parp12 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (39086412..39118349, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (39036411..39068348, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 2 transcripts

Gene: Parp12 ENSMUSG00000038507

Description poly (ADP-ribose) polymerase family, member 12 [Source:MGI Symbol;Acc:MGI:2143990] Gene Synonyms Zc3hdc1 Location Chromosome 6: 39,086,410-39,118,349 reverse strand. GRCm38:CM000999.2 About this gene This gene has 2 transcripts (splice variants), 229 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Parp12-201 ENSMUST00000038398.6 3231 711aa ENSMUSP00000039704.6 Protein coding CCDS20019 Q8BZ20 TSL:1 GENCODE basic APPRIS P1

Parp12-202 ENSMUST00000129916.1 1009 No protein - Retained intron - - TSL:2

51.94 kb Forward strand 39.08Mb 39.09Mb 39.10Mb 39.11Mb 39.12Mb Tbxas1-201 >protein coding 4930599N23Rik-201 >lncRNA (Comprehensive set...

Tbxas1-205 >retained intron

Contigs AC125327.4 > < AC153818.5 Genes (Comprehensive set... < Parp12-201protein coding < Gm42963-201TEC

< Parp12-202retained intron

Regulatory Build

39.08Mb 39.09Mb 39.10Mb 39.11Mb 39.12Mb Reverse strand 51.94 kb

Regulation Legend

CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000038398

< Parp12-201protein coding

Reverse strand 31.94 kb

ENSMUSP00000039... MobiDB lite Low complexity (Seg) Superfamily SSF56399

WWE domain superfamily SMART Zinc finger, CCCH-type Pfam Zinc finger, CCCH-type WWE domain Poly(ADP-ribose) polymerase, catalytic domain

PROSITE profiles Zinc finger, CCCH-type Poly(ADP-ribose) polymerase, catalytic domain

WWE domain PANTHER PTHR45740

PTHR45740:SF9 Gene3D WWE domain superfamily

3.90.228.10 CDD cd01439

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

Variant Legend

missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 711

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

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