https://www.alphaknockout.com Mouse Plac1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Plac1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Plac1 gene (NCBI Reference Sequence: NM_019538 ; Ensembl: ENSMUSG00000061082 ) is located on Mouse chromosome X. 3 exons are identified, with the ATG start codon in exon 3 and the TAA stop codon in exon 3 (Transcript: ENSMUST00000114843). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Plac1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-412J16 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: Female and male mice null for this gene display postnatal lethality with placental abnormalities. Female mice heterozygous for this allele display an enlarged placenta with expansion of the junctional zone. Exon 3 covers 100.0% of the coding region. Start codon is in exon 3, and stop codon is in exon 3. The size of effective cKO region: ~1643 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Plac1 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(6519bp) | A(33.46% 2181) | C(18.87% 1230) | T(25.42% 1657) | G(22.26% 1451) 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. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chrX - 53070832 53073831 3000 browser details YourSeq 106 420 532 3000 97.4% chr1 + 149807556 149807669 114 browser details YourSeq 66 2498 2574 3000 97.3% chr11 + 77628523 77628729 207 browser details YourSeq 64 606 683 3000 94.6% chr10 + 116235882 116236215 334 browser details YourSeq 61 2079 2177 3000 85.9% chr12 - 81540998 81541101 104 browser details YourSeq 61 409 525 3000 78.3% chr10 + 72034704 72034776 73 browser details YourSeq 59 2667 2831 3000 84.1% chr1 - 60660502 60660672 171 browser details YourSeq 58 2723 2855 3000 79.8% chr19 + 46552405 46552528 124 browser details YourSeq 58 2748 2848 3000 91.2% chr1 + 37569156 37569262 107 browser details YourSeq 56 2773 2855 3000 92.6% chr11 - 6222548 6222636 89 browser details YourSeq 56 2723 2849 3000 81.5% chr1 - 171086912 171087032 121 browser details YourSeq 53 2688 2848 3000 92.1% chr10 - 79216210 79216407 198 browser details YourSeq 52 2085 2157 3000 86.2% chr7 + 44640764 44640837 74 browser details YourSeq 52 2754 2839 3000 91.9% chr12 + 51214680 51214772 93 browser details YourSeq 51 416 467 3000 100.0% chr11 + 67751236 67751306 71 browser details YourSeq 51 409 461 3000 98.2% chr1 + 69673134 69673186 53 browser details YourSeq 50 2492 2574 3000 74.2% chr1 - 20301217 20301278 62 browser details YourSeq 50 2087 2179 3000 93.0% chr4 + 94775008 94775238 231 browser details YourSeq 50 2580 2631 3000 98.1% chr11 + 67373162 67373213 52 browser details YourSeq 47 2767 2831 3000 82.9% chr12 - 76848661 76848724 64 Note: The 3000 bp section upstream of Exon 3 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% chrX - 53067313 53070312 3000 browser details YourSeq 430 1347 1872 3000 92.2% chr15 - 69660015 69660587 573 browser details YourSeq 411 1344 1867 3000 92.8% chr1 - 19578718 20053353 474636 browser details YourSeq 402 1347 1835 3000 93.2% chr1 - 144359951 144360796 846 browser details YourSeq 391 1347 1856 3000 90.8% chr8 - 100816195 100817025 831 browser details YourSeq 381 1347 1814 3000 92.5% chr13 + 82739640 82740138 499 browser details YourSeq 377 1352 1856 3000 90.1% chr6 - 68309129 68309643 515 browser details YourSeq 373 1347 1942 3000 91.3% chr15 - 103636914 103637351 438 browser details YourSeq 373 1347 1826 3000 92.5% chr2 + 66278999 66279489 491 browser details YourSeq 343 1349 1986 3000 89.8% chrX + 90180492 90180933 442 browser details YourSeq 334 1344 1941 3000 92.6% chr1 + 4953127 4953754 628 browser details YourSeq 333 1347 1867 3000 91.0% chr1 + 14210466 14210869 404 browser details YourSeq 329 1347 1817 3000 94.4% chr9 - 41710088 41710781 694 browser details YourSeq 328 1347 1861 3000 94.6% chr10 - 111585040 111585869 830 browser details YourSeq 324 1347 1708 3000 95.3% chr11 - 110979329 110979706 378 browser details YourSeq 324 1347 1709 3000 94.8% chr15 + 36032103 36032471 369 browser details YourSeq 323 1347 1709 3000 94.0% chr19 - 21353387 21353748 362 browser details YourSeq 323 1347 1866 3000 90.8% chr11 + 48729600 48729973 374 browser details YourSeq 321 1349 1700 3000 95.8% chr13 - 115633835 115634186 352 browser details YourSeq 321 1347 1709 3000 94.5% chr2 + 150962468 150962835 368 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Plac1 placental specific protein 1 [ Mus musculus (house mouse) ] Gene ID: 56096, updated on 8-Oct-2019 Gene summary Official Symbol Plac1 provided by MGI Official Full Name placental specific protein 1 provided by MGI Primary source MGI:MGI:1926287 See related Ensembl:ENSMUSG00000061082 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 Epcs26; Oosp2l; DXWsu72e Expression Restricted expression toward placenta adult (RPKM 67.5) See more Orthologs human all Genomic context Location: X A5; X 29.31 cM See Plac1 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (53069993..53240110, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (50423179..50467582, complement) Chromosome X - NC_000086.7 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Plac1 ENSMUSG00000061082 Description placental specific protein 1 [Source:MGI Symbol;Acc:MGI:1926287] Gene Synonyms DXWsu72e, Epcs26, Oosp2l Location Chromosome X: 53,069,995-53,240,111 reverse strand. GRCm38:CM001013.2 About this gene This gene has 3 transcripts (splice variants), 62 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 Plac1-202 ENSMUST00000114843.8 1201 173aa ENSMUSP00000110492.2 Protein coding CCDS30128 Q9JI83 TSL:1 GENCODE basic APPRIS P1 Plac1-201 ENSMUST00000074861.8 1041 173aa ENSMUSP00000074404.2 Protein coding CCDS30128 Q9JI83 TSL:1 GENCODE basic APPRIS P1 Plac1-203 ENSMUST00000174390.1 907 173aa ENSMUSP00000133862.1 Protein coding CCDS30128 Q9JI83 TSL:3 GENCODE basic APPRIS P1 190.12 kb Forward strand 53.10Mb 53.15Mb 53.20Mb 53.25Mb Contigs BX649621.5 > BX679673.6 > BX682227.3 > Genes (Comprehensive set... < Plac1-201protein coding < Plac1-202protein coding < Fam122b-201protein coding < Plac1-203protein coding < Fam122b-202protein coding < Fam122b-203protein coding < Fam122b-204lncRNA Regulatory Build 53.10Mb 53.15Mb 53.20Mb 53.25Mb Reverse strand 190.12 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000114843 < Plac1-202protein coding Reverse strand 44.53 kb ENSMUSP00000110... Cleavage site (Sign... PANTHER PLAC1 family Placenta-specific protein 1 Gene3D 2.60.40.3210 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 173 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.