https://www.alphaknockout.com Mouse Ei24 Knockout Project (CRISPR/Cas9) Objective: To create a Ei24 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ei24 gene (NCBI Reference Sequence: NM_001199494 ; Ensembl: ENSMUSG00000062762 ) is located on Mouse chromosome 9. 11 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000115086). Exon 2~7 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: Mice homozygous for a targeted allele do not survive to the neonatal stage. Exon 2 starts from the coding region. Exon 2~7 covers 57.26% of the coding region. The size of effective KO region: ~7817 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 11 Legends Exon of mouse Ei24 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. 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 953 bp section downstream of Exon 7 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(26.45% 529) | C(19.1% 382) | T(34.15% 683) | G(20.3% 406) 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(953bp) | A(28.75% 274) | C(17.21% 164) | T(30.43% 290) | G(23.61% 225) Note: The 953 bp section downstream of Exon 7 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% chr9 - 36793336 36795335 2000 browser details YourSeq 114 12 268 2000 90.9% chr11 - 95920990 95921479 490 browser details YourSeq 106 17 296 2000 90.8% chr12 + 53980126 53980488 363 browser details YourSeq 99 15 294 2000 90.0% chr12 - 86820098 86820376 279 browser details YourSeq 95 12 294 2000 93.6% chr14 + 60970469 61187786 217318 browser details YourSeq 94 15 296 2000 89.9% chr11 + 100507772 100508211 440 browser details YourSeq 88 14 296 2000 94.9% chr18 + 60829439 60829783 345 browser details YourSeq 88 21 294 2000 93.2% chr14 + 49014562 49015081 520 browser details YourSeq 86 13 294 2000 88.4% chr9 - 70674625 70675089 465 browser details YourSeq 79 188 281 2000 97.7% chr6 - 136569584 136569984 401 browser details YourSeq 78 199 294 2000 90.7% chr1 + 51995881 51995976 96 browser details YourSeq 77 14 281 2000 88.9% chr12 - 69138570 69543192 404623 browser details YourSeq 75 218 296 2000 97.5% chr17 + 47393841 47393919 79 browser details YourSeq 73 218 296 2000 97.5% chr16 - 94643023 94643115 93 browser details YourSeq 73 206 292 2000 92.0% chr1 + 132784450 132784536 87 browser details YourSeq 72 224 297 2000 98.7% chrX - 56419117 56419190 74 browser details YourSeq 72 214 296 2000 94.0% chr5 - 35326561 35326644 84 browser details YourSeq 71 218 296 2000 95.0% chr2 - 30308140 30308218 79 browser details YourSeq 71 218 294 2000 96.2% chr16 - 96265410 96265486 77 browser details YourSeq 71 218 296 2000 95.0% chr19 + 26843429 26843507 79 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 953 1 953 953 100.0% chr9 - 36784580 36785532 953 browser details YourSeq 69 503 623 953 82.5% chr1 - 51723972 51724090 119 browser details YourSeq 68 512 626 953 90.5% chr3 + 89852861 89881822 28962 browser details YourSeq 63 512 649 953 86.3% chr1 + 119785646 119786194 549 browser details YourSeq 60 478 569 953 82.7% chrX - 111567762 111567853 92 browser details YourSeq 58 525 634 953 77.8% chrX - 153727146 153727573 428 browser details YourSeq 57 512 664 953 86.9% chrX - 60332910 60333063 154 browser details YourSeq 57 502 623 953 84.4% chr8 - 106943586 106943708 123 browser details YourSeq 55 541 623 953 88.6% chr10 + 59050574 59050656 83 browser details YourSeq 54 539 664 953 83.6% chr12 + 84960080 84960202 123 browser details YourSeq 53 539 626 953 93.5% chr12 - 59136310 59187996 51687 browser details YourSeq 53 497 569 953 87.7% chr4 + 99270697 99270768 72 browser details YourSeq 52 543 632 953 83.9% chr4 + 83531999 83532086 88 browser details YourSeq 52 492 571 953 82.5% chr4 + 44266404 44266483 80 browser details YourSeq 52 550 663 953 90.7% chr17 + 84162292 84162408 117 browser details YourSeq 51 512 578 953 88.6% chr3 - 95912005 95912070 66 browser details YourSeq 51 513 578 953 89.9% chr19 - 44568941 44569005 65 browser details YourSeq 50 520 586 953 87.9% chr10 + 44784408 44784479 72 browser details YourSeq 49 512 578 953 86.6% chr2 - 121723615 121723681 67 browser details YourSeq 49 541 646 953 69.4% chr11 - 9221742 9221843 102 Note: The 953 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Ei24 etoposide induced 2.4 mRNA [ Mus musculus (house mouse) ] Gene ID: 13663, updated on 24-Oct-2019 Gene summary Official Symbol Ei24 provided by MGI Official Full Name etoposide induced 2.4 mRNA provided by MGI Primary source MGI:MGI:108090 See related Ensembl:ENSMUSG00000062762 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 PIG8; AA536736; AI115355 Expression Ubiquitous expression in liver adult (RPKM 47.4), bladder adult (RPKM 40.3) and 28 other tissues See more Orthologs human all Genomic context Location: 9 A4; 9 20.68 cM See Ei24 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (36779153..36797334, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (36586740..36604653, complement) Chromosome 9 - NC_000075.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 11 transcripts Gene: Ei24 ENSMUSG00000062762 Description etoposide induced 2.4 mRNA [Source:MGI Symbol;Acc:MGI:108090] Gene Synonyms PIG8 Location Chromosome 9: 36,779,159-36,797,393 reverse strand. GRCm38:CM001002.2 About this gene This gene has 11 transcripts (splice variants), 223 orthologues, 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 Ei24- ENSMUST00000115086.12 2220 358aa ENSMUSP00000110738.4 Protein CCDS22973 A0A0R4J250 TSL:1 201 coding GENCODE basic Ei24- ENSMUST00000239021.1 2104 358aa ENSMUSP00000159069.1 Protein CCDS22973 - GENCODE basic 211 coding Ei24- ENSMUST00000163192.10 2309 340aa ENSMUSP00000132270.3 Protein - A0A0R4J250 TSL:1 202 coding Q61070 GENCODE basic APPRIS P1 Ei24- ENSMUST00000238932.1 2220 340aa ENSMUSP00000159090.1 Protein - Q61070 GENCODE basic 210 coding APPRIS P1 Ei24- ENSMUST00000184395.8 619 163aa ENSMUSP00000139150.2 Protein - V9GXH2 CDS 3' incomplete 207 coding TSL:3 Ei24- ENSMUST00000184235.1 511 171aa ENSMUSP00000139319.1 Protein - V9GXU0 CDS 5' and 3' 206 coding incomplete TSL:3 Ei24- ENSMUST00000217339.1 1596 No - Retained - - TSL:NA 209 protein intron Ei24- ENSMUST00000185124.1 936 No - Retained - - TSL:2 208 protein intron Ei24- ENSMUST00000183430.1 655 No - Retained - - TSL:3 205 protein intron Ei24- ENSMUST00000183422.1 472 No - Retained - - TSL:2 204 protein intron Ei24- ENSMUST00000183360.1 347 No - lncRNA - - TSL:5 203 protein Page 7 of 9 https://www.alphaknockout.com 38.23 kb Forward strand 36.77Mb 36.78Mb 36.79Mb 36.80Mb Genes Gm26787-201 >lncRNA (Comprehensive set... Contigs < AC155921.2 Genes (Comprehensive set... < Ei24-211protein coding < Ei24-201protein coding < Ei24-210protein coding < Ei24-202protein coding < Ei24-208retained intron < Ei24-207protein coding < Ei24-204retained intron < Ei24-206protein coding < Ei24-205retained intron < Ei24-209retained intron < Ei24-203lncRNA Regulatory Build 36.77Mb 36.78Mb 36.79Mb 36.80Mb Reverse strand 38.23 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000115086 < Ei24-201protein coding Reverse strand 18.23 kb ENSMUSP00000110... Transmembrane heli... Low complexity (Seg) Pfam PF07264 PANTHER Etoposide-induced 2.4 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 358 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.