https://www.alphaknockout.com Mouse Parp10 Knockout Project (CRISPR/Cas9) Objective: To create a Parp10 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Parp10 gene (NCBI Reference Sequence: NM_001163576 ; Ensembl: ENSMUSG00000063268 ) is located on Mouse chromosome 15. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000165738). Exon 1~10 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 1 starts from about 0.03% of the coding region. Exon 1~10 covers 97.47% of the coding region. The size of effective KO region: ~9856 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 8 9 10 Legends Exon of mouse Parp10 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of start codon is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 10 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(27.2% 544) | C(20.15% 403) | T(23.2% 464) | G(29.45% 589) Note: The 2000 bp section upstream of start codon 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(19.9% 398) | C(25.3% 506) | T(21.45% 429) | G(33.35% 667) Note: The 2000 bp section downstream of Exon 10 is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Page 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr15 - 76243236 76245235 2000 browser details YourSeq 43 981 1023 2000 100.0% chr2 + 98599629 98599671 43 browser details YourSeq 39 983 1023 2000 97.6% chrY - 7924541 7924581 41 browser details YourSeq 35 1287 1327 2000 95.0% chr13 - 96254236 96254295 60 browser details YourSeq 29 1289 1325 2000 96.8% chr4 - 119111134 119111173 40 browser details YourSeq 26 1301 1326 2000 100.0% chr18 - 32218083 32218108 26 browser details YourSeq 25 1302 1326 2000 100.0% chr6 - 113934053 113934077 25 browser details YourSeq 25 1302 1326 2000 100.0% chr5 - 117755594 117755618 25 browser details YourSeq 25 1301 1325 2000 100.0% chr2 - 70863646 70863670 25 browser details YourSeq 23 1302 1324 2000 100.0% chr18 - 36536480 36536502 23 browser details YourSeq 21 1302 1322 2000 100.0% chr7 - 6069131 6069151 21 browser details YourSeq 21 1302 1322 2000 100.0% chr14 - 48508291 48508311 21 Note: The 2000 bp section upstream of start codon 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 2000 1 2000 2000 100.0% chr15 - 76231378 76233377 2000 browser details YourSeq 25 1964 1991 2000 84.7% chr1 - 112136521 112136546 26 browser details YourSeq 23 216 243 2000 84.0% chr17 - 31289001 31289026 26 browser details YourSeq 21 1746 1766 2000 100.0% chr18 - 89768996 89769016 21 browser details YourSeq 20 1430 1449 2000 100.0% chr10 - 71298346 71298365 20 browser details YourSeq 20 939 958 2000 100.0% chr1 - 186582429 186582448 20 Note: The 2000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Parp10 poly (ADP-ribose) polymerase family, member 10 [ Mus musculus (house mouse) ] Gene ID: 671535, updated on 10-Oct-2019 Gene summary Official Symbol Parp10 provided by MGI Official Full Name poly (ADP-ribose) polymerase family, member 10 provided by MGI Primary source MGI:MGI:3712326 See related Ensembl:ENSMUSG00000063268 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 ARTD10; PARP-10 Expression Broad expression in thymus adult (RPKM 25.0), adrenal adult (RPKM 24.2) and 20 other tissues See more Orthologs human all Genomic context Location: 15; 15 D3 See Parp10 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (76232995..76243440, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (76063425..76073870, complement) Chromosome 15 - NC_000081.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Parp10 ENSMUSG00000063268 Description poly (ADP-ribose) polymerase family, member 10 [Source:MGI Symbol;Acc:MGI:3712326] Location Chromosome 15: 76,231,174-76,243,441 reverse strand. GRCm38:CM001008.2 About this gene This gene has 6 transcripts (splice variants), 187 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Parp10- ENSMUST00000165738.7 3367 960aa ENSMUSP00000129765.1 Protein coding CCDS49650 Q8CIE4 TSL:1 202 GENCODE basic APPRIS P1 Parp10- ENSMUST00000075689.6 3284 960aa ENSMUSP00000075110.6 Protein coding CCDS49650 Q8CIE4 TSL:1 201 GENCODE basic APPRIS P1 Parp10- ENSMUST00000170226.1 1047 110aa ENSMUSP00000131370.1 Nonsense mediated - - CDS 5' 205 decay incomplete TSL:2 Parp10- ENSMUST00000166151.7 3407 No - Retained intron - - TSL:1 203 protein Parp10- ENSMUST00000170851.1 474 No - Retained intron - - TSL:3 206 protein Parp10- ENSMUST00000169255.1 512 No - lncRNA - - TSL:3 204 protein Page 7 of 9 https://www.alphaknockout.com 32.27 kb Forward strand 76.23Mb 76.24Mb 76.25Mb Genes Grina-203 >protein coding (Comprehensive set... Grina-205 >protein coding Grina-201 >protein coding Grina-202 >protein coding Grina-204 >retained intron Mir6953-201 >miRNA Contigs AC110211.16 > Genes (Comprehensive set... < Plec-209protein coding < Parp10-201protein coding < Plec-206protein coding < Plec-210retained intron < Plec-214protein coding < Parp10-203retained intron < Plec-213protein coding < Parp10-202protein coding < Parp10-205nonsense mediated decay < Parp10-204lncRNA < Parp10-206retained intron Regulatory Build 76.23Mb 76.24Mb 76.25Mb Reverse strand 32.27 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 Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000165738 < Parp10-202protein coding Reverse strand 10.42 kb ENSMUSP00000129... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF56399 Pfam Poly(ADP-ribose) polymerase, catalytic domain PROSITE profiles Poly(ADP-ribose) polymerase, catalytic domain PANTHER PTHR14453:SF53 PTHR14453 Gene3D Nucleotide-binding alpha-beta plait domain superfamily 3.90.228.10 CDD PARP-10, RNA recognition motif 1 and 2 cd01439 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 960 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.