https://www.alphaknockout.com Mouse Pcgf1 Knockout Project (CRISPR/Cas9) Objective: To create a Pcgf1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pcgf1 gene (NCBI Reference Sequence: NM_197992 ; Ensembl: ENSMUSG00000069678 ) is located on Mouse chromosome 6. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000092614). Exon 1~9 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 0.13% of the coding region. Exon 1~9 covers 100.0% of the coding region. The size of effective KO region: ~2333 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 Legends Exon of mouse Pcgf1 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 stop 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. 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(21.3% 426) | C(27.8% 556) | T(26.45% 529) | G(24.45% 489) Note: The 2000 bp section upstream of start codon 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(26.55% 531) | C(23.95% 479) | T(26.25% 525) | G(23.25% 465) Note: The 2000 bp section downstream of stop 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. 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% chr6 + 83076423 83078422 2000 browser details YourSeq 176 209 452 2000 92.3% chr8 - 117013135 117013589 455 browser details YourSeq 175 207 457 2000 93.2% chr4 + 83555387 83555967 581 browser details YourSeq 172 210 452 2000 94.4% chr12 + 81012961 81376357 363397 browser details YourSeq 168 209 448 2000 91.7% chr11 - 75708858 75709246 389 browser details YourSeq 158 191 452 2000 89.5% chr18 - 79691844 79692178 335 browser details YourSeq 152 212 453 2000 91.4% chr7 - 126729893 126730240 348 browser details YourSeq 143 208 456 2000 83.7% chr3 + 120269528 120269698 171 browser details YourSeq 142 208 422 2000 92.4% chr5 - 142986768 142987264 497 browser details YourSeq 140 255 457 2000 92.7% chr16 - 32088844 32089079 236 browser details YourSeq 140 291 457 2000 93.3% chr1 - 16567794 16567963 170 browser details YourSeq 139 206 458 2000 83.5% chr7 + 97113891 97114089 199 browser details YourSeq 139 191 448 2000 83.4% chr4 + 116125277 116125459 183 browser details YourSeq 136 291 458 2000 91.5% chr4 + 29692853 29693022 170 browser details YourSeq 135 191 417 2000 92.0% chr9 - 108650872 108651246 375 browser details YourSeq 135 254 456 2000 92.0% chr15 - 79331160 79331460 301 browser details YourSeq 135 291 453 2000 89.9% chr5 + 121498233 121498392 160 browser details YourSeq 135 254 456 2000 89.5% chr4 + 136018171 136018758 588 browser details YourSeq 134 191 457 2000 82.1% chrX - 95996548 95996710 163 browser details YourSeq 133 291 457 2000 89.0% chr7 - 122118214 122118378 165 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% chr6 + 83080756 83082755 2000 browser details YourSeq 270 594 1662 2000 89.3% chr13 - 46912673 47002822 90150 browser details YourSeq 215 594 1020 2000 91.3% chr14 - 31935196 31936075 880 browser details YourSeq 206 866 1200 2000 91.0% chr6 + 73039231 73039805 575 browser details YourSeq 201 597 1200 2000 82.5% chr9 - 83094638 83095054 417 browser details YourSeq 183 925 1202 2000 89.0% chr3 - 80375732 80376253 522 browser details YourSeq 174 752 1200 2000 83.6% chr11 - 29364031 29364267 237 browser details YourSeq 167 875 1191 2000 89.7% chr3 - 88935460 88936025 566 browser details YourSeq 167 600 1200 2000 79.7% chr1 - 46281666 46282080 415 browser details YourSeq 159 657 1213 2000 80.8% chr19 - 43919827 43920167 341 browser details YourSeq 154 1476 1668 2000 90.6% chr11 - 98860101 98860297 197 browser details YourSeq 154 1488 1674 2000 91.9% chr2 + 25385030 25385218 189 browser details YourSeq 152 1455 1667 2000 91.4% chr3 + 88018249 88018668 420 browser details YourSeq 151 597 1200 2000 79.9% chr14 - 30476683 30477088 406 browser details YourSeq 150 594 1196 2000 79.5% chr6 + 87748423 87748883 461 browser details YourSeq 150 1485 1668 2000 91.3% chr1 + 33723127 33723315 189 browser details YourSeq 149 1479 1668 2000 92.1% chr4 + 151998843 151999200 358 browser details YourSeq 149 1481 1668 2000 90.5% chr4 + 6372474 6372668 195 browser details YourSeq 149 619 1191 2000 78.4% chr3 + 120855983 120856367 385 browser details YourSeq 148 1479 1668 2000 90.3% chr2 - 164072743 164072940 198 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Pcgf1 polycomb group ring finger 1 [ Mus musculus (house mouse) ] Gene ID: 69837, updated on 12-Aug-2019 Gene summary Official Symbol Pcgf1 provided by MGI Official Full Name polycomb group ring finger 1 provided by MGI Primary source MGI:MGI:1917087 See related Ensembl:ENSMUSG00000069678 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 Nspc1; AU024121; 2010002K04Rik Expression Ubiquitous expression in CNS E14 (RPKM 15.0), testis adult (RPKM 12.7) and 28 other tissues See more Orthologs human all Genomic context Location: 6 C3; 6 35.94 cM See Pcgf1 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (83077552..83080855) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (83028384..83030849) Chromosome 6 - NC_000072.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Pcgf1 ENSMUSG00000069678 Description polycomb group ring finger 1 [Source:MGI Symbol;Acc:MGI:1917087] Gene Synonyms 2010002K04Rik, Nspc1 Location Chromosome 6: 83,077,869-83,080,855 forward strand. GRCm38:CM000999.2 About this gene This gene has 8 transcripts (splice variants), 192 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 20 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pcgf1- ENSMUST00000092614.8 866 247aa ENSMUSP00000090277.2 Protein coding CCDS20270 A0A0R4J141 TSL:1 201 GENCODE basic Pcgf1- ENSMUST00000165164.8 918 259aa ENSMUSP00000130614.2 Protein coding - Q8R023 TSL:1 202 GENCODE basic APPRIS P1 Pcgf1- ENSMUST00000177177.7 854 176aa ENSMUSP00000135291.1 Protein coding - H3BK85 TSL:1 207 GENCODE basic Pcgf1- ENSMUST00000176100.1 549 52aa ENSMUSP00000135882.1 Protein coding - H3BLR0 CDS 3' 205 incomplete TSL:3 Pcgf1- ENSMUST00000176089.1 492 108aa ENSMUSP00000135268.1 Protein coding - H3BK63 TSL:5 204 GENCODE basic Pcgf1- ENSMUST00000176027.7 731 106aa ENSMUSP00000135664.1 Nonsense mediated - H3BL61 TSL:5 203 decay Pcgf1- ENSMUST00000204211.1 580 No - Retained intron - - TSL:5 208 protein Pcgf1- ENSMUST00000176372.1 426 No - Retained intron - - TSL:5 206 protein Page 7 of 9 https://www.alphaknockout.com 22.99 kb Forward strand 83.07Mb 83.08Mb 83.09Mb Genes (Comprehensive set... Pcgf1-205 >protein coding Lbx2-201 >protein coding Pcgf1-202 >protein coding Lbx2-202 >lncRNA Pcgf1-201 >protein coding Pcgf1-207 >protein coding Pcgf1-203 >nonsense mediated decay Pcgf1-204 >protein coding Pcgf1-208 >retained intron Pcgf1-206 >retained intron Contigs AC104324.21 > Genes < Tlx2-202protein coding < Gm37092-201TEC < Mir3470a-201miRNA (Comprehensive set... < Tlx2-201protein coding Regulatory Build 83.07Mb 83.08Mb 83.09Mb Reverse strand 22.99 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 RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000092614 2.46 kb Forward strand Pcgf1-201 >protein coding ENSMUSP00000090... Low complexity (Seg) Superfamily SSF57850 SMART Zinc finger, RING-type Pfam PF13923 RAWUL domain PROSITE profiles Zinc finger, RING-type PROSITE patterns Zinc finger, RING-type, conserved site PANTHER PTHR10825:SF29 PTHR10825 Gene3D Zinc finger, RING/FYVE/PHD-type 3.10.20.90 CDD cd16733 cd17081 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 247 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.