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Mouse Armh3 Conditional Knockout Project (CRISPR/Cas9)

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https://www.alphaknockout.com Mouse Armh3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Armh3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Armh3 gene (NCBI Reference Sequence: NM_198296.2 ; Ensembl: ENSMUSG00000039901 ) is located on Mouse chromosome 19. 26 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 26 (Transcript: ENSMUST00000045396). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Armh3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-333H17 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 a knock-out allele exhibit defects in chorion and trophoblast layer formation and complete embryonic lethality during organogenesis. Exon 2 starts from the start codon. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 19703 bp, and the size of intron 2 for 3'-loxP site insertion: 3324 bp. The size of effective cKO region: ~602 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 2 26 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Armh3 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(7102bp) | A(25.53% 1813) | C(20.95% 1488) | T(32.27% 2292) | G(21.25% 1509) 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% chr19 - 45978952 45981951 3000 browser details YourSeq 216 180 886 3000 91.6% chr3 + 157381702 157393517 11816 browser details YourSeq 173 179 516 3000 92.0% chr11 + 82456065 82456477 413 browser details YourSeq 171 188 517 3000 88.1% chr10 + 70062030 70062397 368 browser details YourSeq 167 413 907 3000 91.2% chr10 - 12702650 12901627 198978 browser details YourSeq 166 188 517 3000 88.9% chr15 - 86204991 86205364 374 browser details YourSeq 162 180 528 3000 89.4% chr7 - 121627318 121627711 394 browser details YourSeq 159 180 519 3000 89.7% chr8 - 87320578 87320959 382 browser details YourSeq 159 180 522 3000 94.0% chr19 - 8561950 8562364 415 browser details YourSeq 155 195 904 3000 87.4% chr14 + 27783658 27784387 730 browser details YourSeq 150 182 520 3000 92.1% chr1 - 164847400 164847763 364 browser details YourSeq 150 180 516 3000 84.2% chr1 - 65097007 65097388 382 browser details YourSeq 149 188 522 3000 91.1% chr17 + 48794672 48795059 388 browser details YourSeq 149 179 522 3000 85.8% chr15 + 83909678 83910019 342 browser details YourSeq 146 191 502 3000 91.6% chr7 + 5282287 5282648 362 browser details YourSeq 140 202 515 3000 84.6% chr12 + 71816568 71816931 364 browser details YourSeq 137 180 523 3000 89.1% chrX - 70336841 70337194 354 browser details YourSeq 134 728 896 3000 95.9% chr9 + 100807288 100807468 181 browser details YourSeq 134 180 514 3000 87.0% chr13 + 81224375 81224693 319 browser details YourSeq 133 2507 2651 3000 94.5% chrX - 151185180 151185323 144 Note: The 3000 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 3000 1 3000 3000 100.0% chr19 - 45975350 45978349 3000 browser details YourSeq 403 151 2801 3000 91.1% chr8 - 72205131 72435723 230593 browser details YourSeq 382 154 2590 3000 91.2% chr11 + 20075212 20215176 139965 browser details YourSeq 369 163 2781 3000 90.5% chr1 - 170148467 170183879 35413 browser details YourSeq 169 146 646 3000 94.3% chr2 - 53196072 53196601 530 browser details YourSeq 169 6 323 3000 90.3% chr10 - 56455297 56455607 311 browser details YourSeq 164 161 334 3000 97.7% chr10 - 41301353 41301539 187 browser details YourSeq 162 151 323 3000 97.2% chr4 - 116325812 116325989 178 browser details YourSeq 160 4 321 3000 95.0% chr2 - 4920705 4921128 424 browser details YourSeq 160 153 395 3000 93.9% chr15 - 38534410 38534746 337 browser details YourSeq 160 151 323 3000 96.6% chr8 + 126978390 126978567 178 browser details YourSeq 157 153 323 3000 96.5% chr11 + 116721081 116721259 179 browser details YourSeq 157 161 324 3000 98.2% chr11 + 98122953 98123125 173 browser details YourSeq 157 161 323 3000 98.2% chr10 + 120974297 120974459 163 browser details YourSeq 156 150 321 3000 96.5% chr9 - 21218103 21218463 361 browser details YourSeq 156 162 334 3000 96.5% chr10 - 4345079 4345253 175 browser details YourSeq 155 151 323 3000 95.9% chr10 - 25337304 25337479 176 browser details YourSeq 155 154 322 3000 96.5% chr1 - 131162618 131162787 170 browser details YourSeq 155 161 321 3000 98.2% chr1 - 52882598 52882758 161 browser details YourSeq 155 163 323 3000 98.8% chrX + 8188651 8189191 541 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Armh3 armadillo-like helical domain containing 3 [ Mus musculus (house mouse) ] Gene ID: 71617, updated on 26-Jun-2020 Gene summary Official Symbol Armh3 provided by MGI Official Full Name armadillo-like helical domain containing 3 provided by MGI Primary source MGI:MGI:1918867 See related Ensembl:ENSMUSG00000039901 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 AI431055; AW050122; 9130011E15Rik Expression Ubiquitous expression in heart adult (RPKM 5.9), whole brain E14.5 (RPKM 5.7) and 28 other tissues See more Orthologs human all Genomic context Location: 19; 19 C3 See Armh3 in Genome Data Viewer Exon count: 28 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (45817362..45998632, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (45892634..46072978, complement) Chromosome 19 - NC_000085.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Armh3 ENSMUSG00000039901 Description armadillo-like helical domain containing 3 [Source:MGI Symbol;Acc:MGI:1918867] Gene Synonyms 9130011E15Rik Location Chromosome 19: 45,817,364-45,998,488 reverse strand. GRCm38:CM001012.2 About this gene This gene has 3 transcripts (splice variants), 273 orthologues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Armh3-201 ENSMUST00000045396.8 3676 689aa ENSMUSP00000048454.7 Protein coding CCDS50454 Q6PD19 TSL:5 GENCODE basic APPRIS P1 Armh3-202 ENSMUST00000235131.1 1942 No protein - Processed transcript - - - Armh3-203 ENSMUST00000236730.1 632 No protein - Retained intron - - - Page 6 of 8 https://www.alphaknockout.com 201.12 kb Forward strand 45.85Mb 45.90Mb 45.95Mb 46.00Mb Genes Gm50196-201 >antisense 4930505N22Rik-201 >bidirectional promoter lncRNA (Comprehensive set... 4930505N22Rik-202 >bidirectional promoter lncRNA Hps6-201 >protein coding Contigs AC131185.4 > < AC150685.2 Genes (Comprehensive set... < Kcnip2-202protein coding < Gm6813-201processed pseudogen<e Armh3-202processed transcript < Kcnip2-209protein coding < Armh3-203retained intron < Kcnip2-204retained intron < Kcnip2-203protein coding < Kcnip2-201protein coding < Kcnip2-208protein coding < Kcnip2-210protein coding < Armh3-201protein coding Regulatory Build 45.85Mb 45.90Mb 45.95Mb 46.00Mb Reverse strand 201.12 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Bidirectional promoter lncRNA pseudogene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000045396 < Armh3-201protein coding Reverse strand 181.12 kb ENSMUSP00000048... Low complexity (Seg) Superfamily Armadillo-type fold SMART Domain of unknown function DUF1741 Pfam Domain of unknown function DUF1741 PANTHER Armadillo-like helical domain-containing protein 3-like All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 689 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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