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Mouse Arih2 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Arih2 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Arih2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Arih2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Arih2 gene (NCBI Reference Sequence: NM_011790 ; Ensembl: ENSMUSG00000064145 ) is located on Mouse chromosome 9. 16 exons are identified, with the ATG start codon in exon 3 and the TAA stop codon in exon 16 (Transcript: ENSMUST00000013338). 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 Arih2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-155I7 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: Homozygous inactivation of this gene causes altered dendritic cell physiology, enhanced liver apoptosis, and complete fetal lethality that is partially modified by genetic background. On a mixed genetic background, mice that survive past weaning succumb to a severe multiorgan inflammatory response. Exon 4 starts from about 17.14% 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: 23899 bp, and the size of intron 4 for 3'-loxP site insertion: 2623 bp. The size of effective cKO region: ~568 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 16 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Arih2 Homology arm cKO region loxP site Page 2 of 8 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(7068bp) | A(26.75% 1891) | C(20.19% 1427) | T(31.58% 2232) | G(21.48% 1518) 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 8 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% chr9 - 108620377 108623376 3000 browser details YourSeq 190 419 2251 3000 89.7% chr2 - 115807790 116100777 292988 browser details YourSeq 142 439 1046 3000 81.9% chrX + 53396280 53396645 366 browser details YourSeq 142 410 592 3000 89.5% chr1 + 45525479 45525676 198 browser details YourSeq 140 439 1051 3000 88.8% chr17 + 28253692 28370395 116704 browser details YourSeq 137 406 590 3000 87.5% chr13 + 21741235 21741418 184 browser details YourSeq 136 392 589 3000 86.3% chr2 - 130884911 130885097 187 browser details YourSeq 136 209 590 3000 90.0% chr2 - 10125468 10125988 521 browser details YourSeq 135 406 591 3000 87.9% chr4 - 56853601 56853785 185 browser details YourSeq 135 393 590 3000 81.9% chr3 - 87609511 87609700 190 browser details YourSeq 135 420 590 3000 87.6% chr9 + 110802031 110802199 169 browser details YourSeq 135 406 590 3000 87.4% chr6 + 72540138 72540321 184 browser details YourSeq 134 406 590 3000 86.8% chr5 - 118151722 118151901 180 browser details YourSeq 134 406 588 3000 89.5% chr12 - 72428893 72429085 193 browser details YourSeq 134 439 1048 3000 80.3% chr11 - 39632829 39633196 368 browser details YourSeq 134 405 589 3000 88.9% chr15 + 58396472 58396661 190 browser details YourSeq 132 406 589 3000 88.0% chr2 + 136037788 136037975 188 browser details YourSeq 132 406 590 3000 87.0% chr11 + 57823332 57823516 185 browser details YourSeq 131 414 586 3000 86.0% chr5 - 146812690 146812860 171 browser details YourSeq 131 434 589 3000 90.8% chr9 + 78351436 78351589 154 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% chr9 - 108616809 108619808 3000 browser details YourSeq 210 1430 2175 3000 91.2% chr1 + 136010136 136012789 2654 browser details YourSeq 195 1039 1512 3000 94.2% chr9 + 22208988 22209623 636 browser details YourSeq 178 1990 2256 3000 93.7% chr4 + 41283000 41283410 411 browser details YourSeq 173 1988 2209 3000 92.3% chr6 + 118513047 118513278 232 browser details YourSeq 167 1410 2166 3000 83.1% chr10 + 77671797 77672428 632 browser details YourSeq 166 1989 2256 3000 90.2% chr9 - 20885507 20885823 317 browser details YourSeq 166 1999 2256 3000 91.9% chr8 - 71436703 71436992 290 browser details YourSeq 164 1991 2254 3000 90.2% chr15 - 80766557 80766993 437 browser details YourSeq 162 1990 2256 3000 91.0% chr11 - 75222154 75222475 322 browser details YourSeq 157 1991 2201 3000 86.1% chr5 - 92815766 92815965 200 browser details YourSeq 156 1991 2252 3000 93.3% chr7 - 56081859 56082465 607 browser details YourSeq 156 1991 2210 3000 86.9% chr11 + 33047074 33047263 190 browser details YourSeq 153 1555 2159 3000 82.4% chr14 - 21819277 21819476 200 browser details YourSeq 153 1990 2171 3000 92.8% chr8 + 105940495 105940682 188 browser details YourSeq 153 816 1195 3000 94.8% chr10 + 81159241 81159689 449 browser details YourSeq 150 1988 2178 3000 90.9% chr7 + 29507431 29507629 199 browser details YourSeq 149 1987 2170 3000 89.3% chr14 - 64631967 64632146 180 browser details YourSeq 149 1991 2188 3000 90.4% chr12 - 116513450 116513881 432 browser details YourSeq 149 1989 2175 3000 91.2% chr1 - 105730084 105730272 189 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Arih2 ariadne RBR E3 ubiquitin protein ligase 2 [ Mus musculus (house mouse) ] Gene ID: 23807, updated on 24-Oct-2019 Gene summary Official Symbol Arih2 provided by MGI Official Full Name ariadne RBR E3 ubiquitin protein ligase 2 provided by MGI Primary source MGI:MGI:1344361 See related Ensembl:ENSMUSG00000064145 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 ARI2; UIP48; TRIAD1; AI843547 Expression Ubiquitous expression in testis adult (RPKM 32.8), thymus adult (RPKM 11.1) and 28 other tissues See more Orthologs human all Genomic context Location: 9; 9 F2 See Arih2 in Genome Data Viewer Exon count: 21 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (108602942..108653681, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (108505273..108551711, complement) Chromosome 9 - NC_000075.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 12 transcripts Gene: Arih2 ENSMUSG00000064145 Description ariadne RBR E3 ubiquitin protein ligase 2 [Source:MGI Symbol;Acc:MGI:1344361] Gene Synonyms TRIAD1 Location Chromosome 9: 108,602,942-108,649,386 reverse strand. GRCm38:CM001002.2 About this gene This gene has 12 transcripts (splice variants), 221 orthologues, 9 paralogues, is a member of 1 Ensembl protein family and is associated with 37 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Arih2- ENSMUST00000013338.13 3924 492aa ENSMUSP00000013338.8 Protein coding CCDS23534 Q3TK92 TSL:1 201 Q9Z1K6 GENCODE basic APPRIS P1 Arih2- ENSMUST00000193197.5 570 79aa ENSMUSP00000141911.1 Protein coding - A0A0A6YXA7 CDS 3' 205 incomplete TSL:5 Arih2- ENSMUST00000193190.5 2160 318aa ENSMUSP00000141914.1 Nonsense mediated - A0A0A6YXB0 TSL:5 204 decay Arih2- ENSMUST00000193552.5 793 51aa ENSMUSP00000142102.1 Nonsense mediated - A0A0A6YXR5 CDS 5' 206 decay incomplete TSL:3 Arih2- ENSMUST00000194073.5 780 54aa ENSMUSP00000141364.1 Nonsense mediated - A0A0A6YW24 CDS 5' 209 decay incomplete TSL:3 Arih2- ENSMUST00000193643.1 741 110aa ENSMUSP00000141369.1 Nonsense mediated - A0A0A6YW29 TSL:3 207 decay Arih2- ENSMUST00000193923.1 3664 No - Retained intron - - TSL:NA 208 protein Arih2- ENSMUST00000194290.5 1688 No - Retained intron - - TSL:1 210 protein Arih2- ENSMUST00000195741.5 1518 No - Retained intron - - TSL:3 212 protein Arih2- ENSMUST00000192686.1 650 No - Retained intron - - TSL:3 202 protein Arih2- ENSMUST00000195623.1 435 No - Retained intron - - TSL:3 211 protein Arih2- ENSMUST00000192853.1 263 No - Retained intron - - TSL:3 203 protein Page 6 of 8 https://www.alphaknockout.com 66.44 kb Forward strand 108.60Mb 108.61Mb 108.62Mb 108.63Mb 108.64Mb 108.65Mb Contigs CT571271.12 > Genes (Comprehensive set... < P4htm-201protein coding< Arih2-201protein coding < Gm26298-201miRNA < P4htm-206retained intron< Arih2-208retained intron < Arih2-212retained
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