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Mouse Parp2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Parp2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Parp2 gene (NCBI Reference Sequence: NM_009632 ; Ensembl: ENSMUSG00000036023 ) is located on Mouse chromosome 14. 16 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000036126). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Parp2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-321A24 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 mutant animals are sensitive to gamma radiation. Epithelial crypt degeneration and DNA repair deficiency is apparent following radiation-induced injury.

Exon 5 starts from about 18.13% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 3043 bp, and the size of intron 6 for 3'-loxP site insertion: 769 bp. The size of effective cKO region: ~1058 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 5 6 7 8 9 16 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Parp2 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(7558bp) | A(25.67% 1940) | C(20.88% 1578) | T(31.2% 2358) | G(22.25% 1682)

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% chr14 + 50811621 50814620 3000 browser details YourSeq 462 2044 2899 3000 87.7% chr13 - 32375847 32376504 658 browser details YourSeq 413 2443 2899 3000 95.4% chr5 - 100087229 100087703 475 browser details YourSeq 412 2068 2899 3000 93.7% chr17 + 33516468 33517353 886 browser details YourSeq 411 2044 2879 3000 88.3% chr2 + 36381003 36381824 822 browser details YourSeq 394 2457 2900 3000 95.2% chr10 + 77661501 77661945 445 browser details YourSeq 393 2444 2899 3000 92.8% chr19 - 38808295 38808749 455 browser details YourSeq 392 2145 2899 3000 89.7% chr16 - 97310959 97311421 463 browser details YourSeq 391 2444 2899 3000 93.0% chr4 - 95065200 95065655 456 browser details YourSeq 391 2444 2900 3000 93.6% chr2 - 23408682 23409140 459 browser details YourSeq 391 2441 2899 3000 93.2% chr11 - 70294484 70294944 461 browser details YourSeq 390 2455 2902 3000 94.4% chr10 - 76204706 76205155 450 browser details YourSeq 390 2128 2902 3000 91.2% chr1 - 23424915 23425747 833 browser details YourSeq 389 2470 2902 3000 95.2% chr4 + 122619266 122619714 449 browser details YourSeq 383 2444 2899 3000 92.2% chr3 + 3332734 3333191 458 browser details YourSeq 382 2441 2899 3000 92.9% chr5 - 9004494 9004954 461 browser details YourSeq 381 2451 2899 3000 93.1% chr3 - 117742801 117743251 451 browser details YourSeq 381 2444 2897 3000 93.1% chr2 - 23929039 23929495 457 browser details YourSeq 381 2444 2899 3000 93.2% chr10 + 42673608 42674070 463 browser details YourSeq 379 2444 2903 3000 91.3% chr13 - 96315346 96315799 454

Note: The 3000 bp section upstream of Exon 5 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 + 50815679 50818678 3000 browser details YourSeq 293 2051 2431 3000 89.6% chr4 - 132502032 132502406 375 browser details YourSeq 283 2055 2408 3000 91.9% chr15 - 12366772 12367126 355 browser details YourSeq 280 2050 2407 3000 90.7% chr18 + 77744368 77875350 130983 browser details YourSeq 276 2052 2407 3000 91.4% chr15 + 34148593 34148995 403 browser details YourSeq 271 2051 2399 3000 91.2% chr11 - 104323302 104323886 585 browser details YourSeq 265 2056 2407 3000 91.1% chr19 - 5329691 5330121 431 browser details YourSeq 264 2079 2407 3000 91.3% chr11 + 88988256 88988933 678 browser details YourSeq 253 2062 2408 3000 92.7% chr16 - 18494333 18494798 466 browser details YourSeq 251 2073 2403 3000 91.0% chr19 - 37462923 37463258 336 browser details YourSeq 244 2069 2391 3000 91.6% chr5 + 113835400 113835937 538 browser details YourSeq 234 2074 2391 3000 91.8% chr5 + 77276725 77277046 322 browser details YourSeq 229 2047 2407 3000 84.4% chr11 + 120211267 120211618 352 browser details YourSeq 228 2067 2399 3000 89.4% chr1 + 168442107 168442455 349 browser details YourSeq 227 2077 2373 3000 92.3% chr9 + 120699169 120699721 553 browser details YourSeq 225 2067 2361 3000 90.4% chr19 + 7250604 7250896 293 browser details YourSeq 221 2082 2407 3000 85.8% chr17 - 32132904 32133226 323 browser details YourSeq 220 2103 2407 3000 88.8% chr3 - 94972289 94972615 327 browser details YourSeq 220 2080 2407 3000 88.1% chr17 - 56223489 56223803 315 browser details YourSeq 217 2088 2408 3000 89.2% chr15 - 97427698 97428034 337

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Parp2 poly (ADP-ribose) polymerase family, member 2 [ Mus musculus (house mouse) ] Gene ID: 11546, updated on 10-Oct-2019

Gene summary

Official Symbol Parp2 provided by MGI Official Full Name poly (ADP-ribose) polymerase family, member 2 provided by MGI Primary source MGI:MGI:1341112 See related Ensembl:ENSMUSG00000036023 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 ARTD2; Adprt2; C78626; PARP-2; ADPRT-2; Adprtl2; mPARP-2; Aspartl2; pADPRT-2 Expression Ubiquitous expression in CNS E11.5 (RPKM 20.7), liver E14 (RPKM 15.1) and 25 other tissues See more Orthologs human all

Genomic context

Location: 14 26.25 cM; 14 C1 See Parp2 in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (50807897..50821301)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (51427621..51440975)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 8 transcripts

Gene: Parp2 ENSMUSG00000036023

Description poly (ADP-ribose) polymerase family, member 2 [Source:MGI Symbol;Acc:MGI:1341112] Gene Synonyms Adprt2, Adprtl2, Aspartl2, C78626, PARP-2 Location Chromosome 14: 50,807,841-50,821,301 forward strand. GRCm38:CM001007.2 About this gene This gene has 8 transcripts (splice variants), 172 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Parp2- ENSMUST00000036126.6 1894 559aa ENSMUSP00000048877.5 Protein coding CCDS27025 O88554 TSL:1 201 GENCODE basic APPRIS P1

Parp2- ENSMUST00000227810.1 737 42aa ENSMUSP00000153963.1 Nonsense mediated - A0A2I3BPY1 CDS 5' 204 decay incomplete

Parp2- ENSMUST00000228672.1 2173 No - Retained intron - - - 206 protein

Parp2- ENSMUST00000226880.1 1756 No - Retained intron - - - 202 protein

Parp2- ENSMUST00000228624.1 503 No - Retained intron - - - 205 protein

Parp2- ENSMUST00000228872.1 423 No - Retained intron - - - 208 protein

Parp2- ENSMUST00000228833.1 220 No - Retained intron - - - 207 protein

Parp2- ENSMUST00000227705.1 342 No - lncRNA - - - 203 protein

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33.46 kb Forward strand 50.80Mb 50.81Mb 50.82Mb 50.83Mb Genes Parp2-201 >protein coding (Comprehensive set...

Parp2-202 >retained intron Parp2-205 >retained intron

Parp2-203 >lncRNA Parp2-204 >nonsense mediated decay

Parp2-206 >retained intron

Parp2-207 >retained intron

Parp2-208 >retained intron

Contigs < AC027184.15 Genes < Ccnb1ip1-202protein coding < Tep1-201protein coding (Comprehensive set...

< Rpph1-201ribozyme < Tep1-202protein coding

< Tep1-203nonsense mediated decay

< Tep1-207nonsense mediated decay

< Tep1-208retained intron

< Tep1-206retained intron

Regulatory Build

50.80Mb 50.81Mb 50.82Mb 50.83Mb Reverse strand 33.46 kb

Regulation Legend CTCF 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: ENSMUST00000036126

13.46 kb Forward strand

Parp2-201 >protein coding

ENSMUSP00000048... PDB-ENSP mappings MobiDB lite Superfamily WGR domain superfamily Poly(ADP-ribose) polymerase, regulatory domain superfamily

SSF56399 SMART WGR domain Pfam WGR domain Poly(ADP-ribose) polymerase, catalytic domain

Poly(ADP-ribose) polymerase, regulatory domain PROSITE profiles Poly(ADP-ribose) polymerase, catalytic domain

Poly(ADP-ribose) polymerase, regulatory domain PANTHER PTHR10459:SF60

PTHR10459 Gene3D WGR domain superfamily 3.90.228.10

Poly(ADP-ribose) polymerase, regulatory domain superfamily CDD cd08003 cd01437

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 559

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

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