https://www.alphaknockout.com Mouse Ppef1 Knockout Project (CRISPR/Cas9) Objective: To create a Ppef1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ppef1 gene (NCBI Reference Sequence: NM_011147 ; Ensembl: ENSMUSG00000062168 ) is located on Mouse chromosome X. 16 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 16 (Transcript: ENSMUST00000071204). Exon 2~4 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: Homozygous null female and hemizygous null male mice are viable, fertile and display no overt abnormalities. Exon 2 starts from about 2.41% of the coding region. Exon 2~4 covers 18.41% of the coding region. The size of effective KO region: ~10105 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 16 Legends Exon of mouse Ppef1 Knockout region Page 2 of 8 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 Exon 2 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 4 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 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(26.6% 532) | C(21.85% 437) | T(32.1% 642) | G(19.45% 389) Note: The 2000 bp section upstream of Exon 2 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(25.35% 507) | C(17.15% 343) | T(41.9% 838) | G(15.6% 312) Note: The 2000 bp section downstream of Exon 4 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 8 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% chrX - 160693615 160695614 2000 browser details YourSeq 345 47 460 2000 92.9% chr10 + 63563113 63563531 419 browser details YourSeq 344 48 460 2000 90.9% chrX - 20524959 20525361 403 browser details YourSeq 344 66 455 2000 94.2% chr1 - 189592053 189592442 390 browser details YourSeq 342 45 460 2000 91.4% chr14 - 103006245 103006661 417 browser details YourSeq 339 47 461 2000 90.1% chr5 + 72819370 72819782 413 browser details YourSeq 339 47 456 2000 92.5% chr1 + 150376871 150377281 411 browser details YourSeq 338 66 456 2000 93.4% chr1 + 43638403 43638812 410 browser details YourSeq 337 48 456 2000 91.1% chrX + 113388176 113388582 407 browser details YourSeq 336 47 460 2000 89.9% chr6 - 74629636 74630039 404 browser details YourSeq 336 68 456 2000 93.3% chr16 - 59705165 59705553 389 browser details YourSeq 336 46 456 2000 90.3% chr1 + 16403509 16403911 403 browser details YourSeq 335 45 456 2000 92.3% chr10 - 9217848 9218270 423 browser details YourSeq 335 45 456 2000 89.9% chr12 + 28354431 28354835 405 browser details YourSeq 334 68 456 2000 93.1% chrX - 10092007 10092396 390 browser details YourSeq 334 52 456 2000 90.2% chr4 + 85541322 85541718 397 browser details YourSeq 333 49 456 2000 90.0% chr5 - 5813440 5813839 400 browser details YourSeq 332 47 456 2000 90.7% chr3 - 68978834 68979244 411 browser details YourSeq 332 48 452 2000 92.1% chr2 - 90798654 90799060 407 browser details YourSeq 332 53 456 2000 92.0% chr18 - 74915569 74915977 409 Note: The 2000 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 2000 1 2000 2000 100.0% chrX - 160683170 160685169 2000 browser details YourSeq 151 506 670 2000 96.4% chr11 + 94599432 94599609 178 browser details YourSeq 149 506 673 2000 94.7% chr8 - 85006434 85006601 168 browser details YourSeq 149 514 686 2000 95.2% chr11 - 63433813 63433988 176 browser details YourSeq 147 506 684 2000 90.2% chr5 + 131037140 131037316 177 browser details YourSeq 147 510 682 2000 94.1% chr18 + 12169974 12170146 173 browser details YourSeq 146 507 690 2000 92.0% chr2 - 92900791 92900984 194 browser details YourSeq 146 508 686 2000 93.5% chr15 - 103249746 103249927 182 browser details YourSeq 146 511 673 2000 95.1% chr9 + 72378238 72378401 164 browser details YourSeq 146 463 670 2000 94.6% chr4 + 48535228 48535485 258 browser details YourSeq 146 506 672 2000 94.0% chr1 + 91461179 91461366 188 browser details YourSeq 145 506 666 2000 95.7% chr19 - 4694940 4695137 198 browser details YourSeq 143 500 666 2000 95.0% chr1 - 56798758 56798935 178 browser details YourSeq 142 522 688 2000 93.3% chr3 - 127582033 127582204 172 browser details YourSeq 141 518 689 2000 93.8% chr2 - 59500937 59501113 177 browser details YourSeq 141 510 673 2000 91.4% chr12 + 83704558 83704719 162 browser details YourSeq 139 518 670 2000 95.5% chr16 + 31752739 31752891 153 browser details YourSeq 139 518 673 2000 94.9% chr13 + 29863861 29864019 159 browser details YourSeq 139 521 676 2000 94.9% chr10 + 22711434 22711595 162 browser details YourSeq 138 518 670 2000 95.5% chr19 - 38842373 38842536 164 Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Ppef1 protein phosphatase with EF hand calcium-binding domain 1 [ Mus musculus (house mouse) ] Gene ID: 237178, updated on 3-Sep-2019 Gene summary Official Symbol Ppef1 provided by MGI Official Full Name protein phosphatase with EF hand calcium-binding domain 1 provided by MGI Primary source MGI:MGI:1097157 See related Ensembl:ENSMUSG00000062168 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 PPEF-1 Expression Biased expression in testis adult (RPKM 1.6), CNS E14 (RPKM 0.1) and 1 other tissue See more Orthologs human all Genomic context Location: X F4; X 73.95 cM See Ppef1 in Genome Data Viewer Exon count: 20 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (160622419..160751290, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (157061026..157157904, complement) Chromosome X - NC_000086.7 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Ppef1 ENSMUSG00000062168 Description protein phosphatase with EF hand calcium-binding domain 1 [Source:MGI Symbol;Acc:MGI:1097157] Gene Synonyms Dres10, PPEF 1, PPEF-1 Location Chromosome X: 160,623,094-160,735,765 reverse strand. GRCm38:CM001013.2 About this gene This gene has 3 transcripts (splice variants), 215 orthologues, 12 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ppef1-201 ENSMUST00000071204.11 2483 650aa ENSMUSP00000071191.5 Protein coding CCDS41200 O35655 TSL:2 GENCODE basic APPRIS P1 Ppef1-202 ENSMUST00000135856.1 1064 237aa ENSMUSP00000123408.1 Protein coding - Q3UQZ5 CDS 3' incomplete TSL:1 Ppef1-203 ENSMUST00000136888.1 252 56aa ENSMUSP00000121692.1 Protein coding - B7ZCG2 CDS 3' incomplete TSL:5 132.67 kb Forward strand 160.62Mb 160.64Mb 160.66Mb 160.68Mb 160.70Mb 160.72Mb 160.74Mb Genes Gm15244-201 >processed pseudogene (Comprehensive set... Contigs AL732450.5 > AL669939.9 > Genes (Comprehensive set... < Ppef1-201protein coding < Ppef1-202protein coding < Ppef1-203protein coding < Gm15242-201processed pseudogene Regulatory Build 160.62Mb 160.64Mb 160.66Mb 160.68Mb 160.70Mb 160.72Mb 160.74Mb Reverse strand 132.67 kb Regulation Legend CTCF Open Chromatin Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000071204 < Ppef1-201protein coding Reverse strand 96.88 kb ENSMUSP00000071... MobiDB lite Low complexity (Seg) Superfamily SSF56300 EF-hand domain pair SMART Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase IQ motif, EF-hand binding site EF-hand domain Prints Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase Pfam PPP domain Calcineurin-like phosphoesterase domain, ApaH type EF-hand domain PROSITE profiles EF-hand domain PROSITE patterns Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase EF-Hand 1, calcium-binding site PIRSF Serine/threonine-protein phosphatase with EF-hands PANTHER PTHR45668:SF1 PTHR45668 Gene3D Metallo-dependent phosphatase-like 1.10.238.10 CDD cd07420 EF-hand domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 650 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.