https://www.alphaknockout.com Mouse Casp8ap2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Casp8ap2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Casp8ap2 gene (NCBI Reference Sequence: NM_011997 ; Ensembl: ENSMUSG00000028282 ) is located on Mouse chromosome 4. 11 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000029950). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Casp8ap2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-75H14 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: Mice homozygous for disruption of this gene die before implantation. Exon 4 starts from about 2.12% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 819 bp, and the size of intron 4 for 3'-loxP site insertion: 639 bp. The size of effective cKO region: ~601 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 gRNA region 5' gRNA region 3' 1 3 4 5 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Casp8ap2 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. 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7101bp) | A(26.6% 1889) | C(19.39% 1377) | T(33.71% 2394) | G(20.29% 1441) 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% chr4 + 32627817 32630816 3000 browser details YourSeq 200 233 2212 3000 94.4% chr9 + 119785036 120133031 347996 browser details YourSeq 198 217 2211 3000 94.7% chr1 - 36716383 36848910 132528 browser details YourSeq 195 1897 2209 3000 91.2% chr9 + 101083164 101083802 639 browser details YourSeq 167 1919 2218 3000 89.6% chr8 + 33568937 33569494 558 browser details YourSeq 163 1900 2199 3000 89.8% chr2 - 155344489 155345098 610 browser details YourSeq 156 1892 2173 3000 94.4% chr2 + 154395889 154396541 653 browser details YourSeq 143 1874 2052 3000 92.1% chrX + 101777446 101777622 177 browser details YourSeq 142 1873 2044 3000 89.4% chr3 - 86018030 86018191 162 browser details YourSeq 139 1875 2052 3000 87.8% chr1 - 130617574 130617737 164 browser details YourSeq 136 1890 2053 3000 91.9% chr11 - 100987614 100987789 176 browser details YourSeq 136 1914 2314 3000 96.0% chr4 + 44224587 44225157 571 browser details YourSeq 134 1882 2052 3000 90.4% chr3 - 34633244 34633423 180 browser details YourSeq 133 1895 2212 3000 84.5% chr1 - 64958186 64958359 174 browser details YourSeq 132 1895 2200 3000 91.3% chr12 - 86869234 86869800 567 browser details YourSeq 132 1899 2052 3000 95.2% chr10 - 128022502 128022657 156 browser details YourSeq 132 1893 2048 3000 95.3% chr7 + 30048749 30049296 548 browser details YourSeq 131 1882 2044 3000 88.7% chr8 - 114901617 114901770 154 browser details YourSeq 131 1874 2050 3000 86.5% chr3 - 116580897 116581059 163 browser details YourSeq 130 1888 2044 3000 89.8% chr8 + 69211933 69212083 151 Note: The 3000 bp section upstream of Exon 4 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% chr4 + 32631418 32634417 3000 browser details YourSeq 259 810 1550 3000 80.9% chr9 + 57843631 57844518 888 browser details YourSeq 235 1419 2552 3000 91.3% chr11 - 6205222 6459959 254738 browser details YourSeq 203 862 1439 3000 86.1% chr10 - 4200403 4351547 151145 browser details YourSeq 186 840 1216 3000 87.2% chr13 - 28892793 28893214 422 browser details YourSeq 183 880 1216 3000 84.8% chr7 - 64407649 64408020 372 browser details YourSeq 167 807 1542 3000 81.0% chrX - 100712861 100713499 639 browser details YourSeq 161 805 1214 3000 88.9% chrX + 100961971 100962437 467 browser details YourSeq 159 810 1195 3000 89.3% chr2 + 61552959 61553386 428 browser details YourSeq 146 863 1216 3000 89.4% chr6 + 112977397 112977808 412 browser details YourSeq 142 2424 2768 3000 84.9% chr1 + 49465745 49465989 245 browser details YourSeq 133 879 1163 3000 88.9% chr5 - 144096500 144096840 341 browser details YourSeq 129 2428 2573 3000 94.6% chr4 + 5397236 5397382 147 browser details YourSeq 128 2427 2575 3000 93.3% chr7 - 141575782 141575930 149 browser details YourSeq 128 2428 2576 3000 94.0% chr15 - 17199210 17199364 155 browser details YourSeq 128 2428 2575 3000 94.0% chr19 + 32998579 32998728 150 browser details YourSeq 127 2427 2575 3000 93.3% chr5 - 64725548 64725705 158 browser details YourSeq 127 2424 2573 3000 92.7% chr9 + 15289739 15289889 151 browser details YourSeq 126 879 1216 3000 86.7% chr7 - 29772136 29772535 400 browser details YourSeq 125 2427 2575 3000 93.3% chrX - 36419814 36419974 161 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Casp8ap2 caspase 8 associated protein 2 [ Mus musculus (house mouse) ] Gene ID: 26885, updated on 10-Oct-2019 Gene summary Official Symbol Casp8ap2 provided by MGI Official Full Name caspase 8 associated protein 2 provided by MGI Primary source MGI:MGI:1349399 See related Ensembl:ENSMUSG00000028282 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 FLASH; D4Ertd659e Expression Broad expression in CNS E11.5 (RPKM 6.6), placenta adult (RPKM 3.7) and 17 other tissues See more Orthologs human all Genomic context Location: 4 A5; 4 14.27 cM See Casp8ap2 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (32615470..32653271) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (32702448..32740240) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Casp8ap2 ENSMUSG00000028282 Description caspase 8 associated protein 2 [Source:MGI Symbol;Acc:MGI:1349399] Gene Synonyms D4Ertd659e, FLASH Location Chromosome 4: 32,615,451-32,653,265 forward strand. GRCm38:CM000997.2 About this gene This gene has 5 transcripts (splice variants), 197 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 Casp8ap2-201 ENSMUST00000029950.9 6799 1962aa ENSMUSP00000029950.3 Protein coding CCDS18016 Q9WUF3 TSL:1 GENCODE basic APPRIS P2 Casp8ap2-205 ENSMUST00000178925.7 6557 1962aa ENSMUSP00000136016.1 Protein coding CCDS18016 Q9WUF3 TSL:5 GENCODE basic APPRIS P2 Casp8ap2-202 ENSMUST00000108178.1 1189 190aa ENSMUSP00000103813.1 Protein coding - B1AX75 TSL:5 GENCODE basic APPRIS ALT2 Casp8ap2-204 ENSMUST00000127619.1 595 No protein - lncRNA - - TSL:3 Casp8ap2-203 ENSMUST00000124278.7 300 No protein - lncRNA - - TSL:5 57.81 kb Forward strand 32.61Mb 32.62Mb 32.63Mb 32.64Mb 32.65Mb 32.66Mb Genes (Comprehensive set... Gm11933-201 >processed pseudogene Casp8ap2-202 >protein coding Mdn1-201 >protein coding Casp8ap2-203 >lncRNA Mdn1-211 >protein coding Casp8ap2-204 >lncRNA Mdn1-210 >lncRNA Casp8ap2-205 >protein coding Casp8ap2-201 >protein coding Contigs AL831746.5 > AL805973.6 > Genes < Gm11936-201processed pseudogene (Comprehensive set... Regulatory Build 32.61Mb 32.62Mb 32.63Mb 32.64Mb 32.65Mb 32.66Mb Reverse strand 57.81 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000029950 37.79 kb Forward strand Casp8ap2-201 >protein coding ENSMUSP00000029... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) PANTHER CASP8-associated protein 2 Gene3D 1.10.10.60 CDD cd12202 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe insertion inframe deletion missense variant synonymous variant Scale bar 0 200 400 600 800 1000 1200 1400 1600 1962 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.