https://www.alphaknockout.com Mouse Cped1 Knockout Project (CRISPR/Cas9) Objective: To create a Cped1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cped1 gene (NCBI Reference Sequence: NM_001081351 ; Ensembl: ENSMUSG00000062980 ) is located on Mouse chromosome 6. 22 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 22 (Transcript: ENSMUST00000115383). Exon 2 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 2 starts from about 8.12% of the coding region. Exon 2 covers 5.98% of the coding region. The size of effective KO region: ~184 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 gRNA region 5' gRNA region 3' 1 2 22 Legends Exon of mouse Cped1 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 Exon 2 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 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. 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(25.65% 513) | C(21.55% 431) | T(28.2% 564) | G(24.6% 492) 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(29.7% 594) | C(19.2% 384) | T(30.8% 616) | G(20.3% 406) Note: The 2000 bp section downstream 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. 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 + 22014904 22016903 2000 browser details YourSeq 305 6 761 2000 88.3% chr11 + 50845713 50846728 1016 browser details YourSeq 295 61 780 2000 83.1% chr1 - 125754881 125755643 763 browser details YourSeq 288 8 757 2000 85.7% chr1 - 157628160 157628903 744 browser details YourSeq 267 6 582 2000 84.5% chr13 + 96004138 96005082 945 browser details YourSeq 265 11 766 2000 87.1% chr14 + 122994096 122994893 798 browser details YourSeq 254 249 785 2000 85.6% chr1 - 139323774 139324339 566 browser details YourSeq 247 42 726 2000 82.5% chr4 + 21629387 21630000 614 browser details YourSeq 234 6 775 2000 82.9% chr3 - 158320314 158612318 292005 browser details YourSeq 234 61 766 2000 84.1% chr9 + 95419202 95419974 773 browser details YourSeq 233 12 768 2000 84.8% chr16 + 11801774 11802873 1100 browser details YourSeq 229 1 703 2000 75.5% chrX + 161462538 161463195 658 browser details YourSeq 227 38 756 2000 87.6% chr5 - 73443013 73443725 713 browser details YourSeq 227 8 726 2000 78.0% chr13 - 96767861 96768490 630 browser details YourSeq 224 136 764 2000 86.5% chr6 - 55320595 55321326 732 browser details YourSeq 224 10 766 2000 88.5% chr7 + 73039876 73040724 849 browser details YourSeq 218 43 766 2000 79.3% chr8 - 127752564 127753221 658 browser details YourSeq 216 125 749 2000 77.0% chr17 - 24026745 24027345 601 browser details YourSeq 216 3 761 2000 80.7% chr11 - 30244722 30245403 682 browser details YourSeq 216 38 770 2000 87.3% chr11 + 43516421 43517368 948 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% chr6 + 22017088 22019087 2000 browser details YourSeq 24 1803 1827 2000 100.0% chr7 - 78032026 78032051 26 browser details YourSeq 23 122 144 2000 100.0% chr15 - 102063870 102063892 23 browser details YourSeq 23 71 93 2000 100.0% chr18 + 81043926 81043948 23 browser details YourSeq 22 71 92 2000 100.0% chr6 - 43695866 43695887 22 browser details YourSeq 22 72 95 2000 87.0% chr18 - 67601709 67601731 23 browser details YourSeq 22 48 69 2000 100.0% chr12 + 10812695 10812716 22 browser details YourSeq 20 73 92 2000 100.0% chr11 + 53719232 53719251 20 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Cped1 cadherin-like and PC-esterase domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 214642, updated on 12-Aug-2019 Gene summary Official Symbol Cped1 provided by MGI Official Full Name cadherin-like and PC-esterase domain containing 1 provided by MGI Primary source MGI:MGI:2444814 See related Ensembl:ENSMUSG00000062980 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as AI552584; 6720481P07; A430107O13Rik Expression Broad expression in subcutaneous fat pad adult (RPKM 13.7), bladder adult (RPKM 10.2) and 19 other tissues See more Orthologs human all Genomic context Location: 6; 6 A3.1 See Cped1 in Genome Data Viewer Exon count: 24 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (21985710..22256407) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (21935910..22205606) Chromosome 6 - NC_000072.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Cped1 ENSMUSG00000062980 Description cadherin-like and PC-esterase domain containing 1 [Source:MGI Symbol;Acc:MGI:2444814] Gene Synonyms A430107O13Rik Location Chromosome 6: 21,985,916-22,256,404 forward strand. GRCm38:CM000999.2 About this gene This gene has 8 transcripts (splice variants), 180 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cped1- ENSMUST00000115383.8 5690 1026aa ENSMUSP00000111041.2 Protein coding CCDS39435 B2RX70 TSL:1 202 GENCODE basic APPRIS P2 Cped1- ENSMUST00000137437.5 2484 828aa ENSMUSP00000119808.2 Protein coding - E9Q7L8 CDS 5' and 3' 203 incomplete TSL:1 APPRIS ALT2 Cped1- ENSMUST00000115382.7 2002 473aa ENSMUSP00000111040.1 Protein coding - D3YUQ2 TSL:5 201 GENCODE basic APPRIS ALT2 Cped1- ENSMUST00000153922.7 3520 162aa ENSMUSP00000138562.1 Nonsense mediated - S4R2A1 TSL:1 206 decay Cped1- ENSMUST00000154734.1 643 No - Retained intron - - TSL:3 207 protein Cped1- ENSMUST00000141064.1 1794 No - lncRNA - - TSL:1 204 protein Cped1- ENSMUST00000151315.4 645 No - lncRNA - - TSL:3 205 protein Cped1- ENSMUST00000156621.1 399 No - lncRNA - - TSL:5 208 protein Page 7 of 9 https://www.alphaknockout.com 290.49 kb Forward strand 22.0Mb 22.1Mb 22.2Mb Genes (Comprehensive set... Ing3-201 >protein coding Cped1-203 >protein coding Cped1-202 >protein coding Cped1-206 >nonsense mediated decay Cped1-207 >retained intron Cped1-204 >lncRNA Cped1-208 >lncRNA Cped1-201 >protein coding Cped1-205 >lncRNA Contigs AC117213.3 > AC133955.4 > Genes < Gm42573-201processed pseudogene (Comprehensive set... Regulatory Build 22.0Mb 22.1Mb 22.2Mb Reverse strand 290.49 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000115383 270.49 kb Forward strand Cped1-202 >protein coding ENSMUSP00000111... Transmembrane heli... Low complexity (Seg) Pfam Cadherin-like beta sandwich domain PANTHER PTHR14776 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe insertion missense variant splice region variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 900 1026 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.