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Mouse Kmo Conditional Knockout Project (CRISPR/Cas9)

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Mouse Kmo Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Kmo Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Kmo conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Kmo gene (NCBI Reference Sequence: NM_133809 ; Ensembl: ENSMUSG00000039783 ) is located on Mouse chromosome 1. 15 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 15 (Transcript: ENSMUST00000040250). Exon 6~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Kmo gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-129O14 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 lack kynurenine 3-monooxygenase activity and altered levels of several tryptophan metabolites. Mice homozygous for another null allele exhibit increased LPS-induced depressive behaviors and altered kynurenine metabolism. Exon 6 starts from about 25.19% of the coding region. The knockout of Exon 6~7 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 3506 bp, and the size of intron 7 for 3'-loxP site insertion: 1247 bp. The size of effective cKO region: ~2705 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 5' gRNA region gRNA region 3' 1 6 7 8 15 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Kmo 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(9205bp) | A(27.01% 2486) | C(20.88% 1922) | T(31.21% 2873) | G(20.9% 1924) 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% chr1 + 175643864 175646863 3000 browser details YourSeq 188 681 1268 3000 88.9% chr10 - 118586635 118681729 95095 browser details YourSeq 166 552 1295 3000 89.1% chr16 - 24111761 24112504 744 browser details YourSeq 165 674 1230 3000 90.2% chr3 + 30781658 30782268 611 browser details YourSeq 164 600 1246 3000 88.8% chr3 - 35674649 35675322 674 browser details YourSeq 155 811 1247 3000 88.0% chr9 - 86364038 86364498 461 browser details YourSeq 145 738 1247 3000 88.1% chr7 - 135321115 135321635 521 browser details YourSeq 142 672 1266 3000 87.5% chrX - 136623556 136624180 625 browser details YourSeq 136 844 1266 3000 87.4% chr19 + 32802233 32802681 449 browser details YourSeq 134 672 1161 3000 91.8% chr18 - 83511910 83934632 422723 browser details YourSeq 127 659 1114 3000 91.1% chr3 - 106011458 106011915 458 browser details YourSeq 123 672 1158 3000 85.7% chr11 - 55307665 55308164 500 browser details YourSeq 122 641 1265 3000 90.8% chr10 - 114478366 114479057 692 browser details YourSeq 122 728 1133 3000 86.4% chr17 + 63881457 63881886 430 browser details YourSeq 121 676 1136 3000 81.9% chr2 + 143797944 143798351 408 browser details YourSeq 113 674 1189 3000 81.4% chr1 - 191463373 191463862 490 browser details YourSeq 110 954 1266 3000 85.0% chr7 + 65117843 65118176 334 browser details YourSeq 104 845 1305 3000 82.9% chr14 - 45170424 45170872 449 browser details YourSeq 104 496 976 3000 84.3% chr14 - 36807605 36808075 471 browser details YourSeq 103 493 980 3000 84.7% chrX + 138790323 138790785 463 Note: The 3000 bp section upstream of Exon 6 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% chr1 + 175649569 175652568 3000 browser details YourSeq 301 217 560 3000 93.6% chr10 + 23260598 23260939 342 browser details YourSeq 179 1 220 3000 91.0% chr6 - 135264779 135264995 217 browser details YourSeq 85 2914 3000 3000 98.9% chr11 - 71970822 71970908 87 browser details YourSeq 84 2913 3000 3000 97.8% chr19 - 16553948 16554035 88 browser details YourSeq 84 2913 3000 3000 97.8% chr18 - 54584426 54584513 88 browser details YourSeq 84 2913 3000 3000 97.8% chr13 - 3383806 3383893 88 browser details YourSeq 84 2913 3000 3000 97.8% chr17 + 36246817 36246904 88 browser details YourSeq 83 2914 3000 3000 97.8% chr9 - 8174458 8174544 87 browser details YourSeq 83 2914 3000 3000 97.8% chr4 - 48678870 48678956 87 browser details YourSeq 83 2913 3000 3000 97.8% chrX + 155723785 155723924 140 browser details YourSeq 83 2914 3000 3000 97.8% chr6 + 51555300 51555386 87 browser details YourSeq 83 2914 3000 3000 97.8% chr4 + 141798449 141798535 87 browser details YourSeq 83 2914 3000 3000 97.8% chr11 + 41256028 41256114 87 browser details YourSeq 83 2913 3000 3000 97.8% chr11 + 11111955 11112045 91 browser details YourSeq 82 2913 3000 3000 96.6% chr4 - 34369306 34369393 88 browser details YourSeq 82 2913 3000 3000 96.6% chr3 - 61825794 61825881 88 browser details YourSeq 82 2913 3000 3000 96.6% chr14 - 61479849 61479936 88 browser details YourSeq 82 2913 3000 3000 96.6% chr12 - 50025994 50026081 88 browser details YourSeq 82 2913 3000 3000 96.6% chr3 + 47383999 47384086 88 Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Kmo kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) [ Mus musculus (house mouse) ] Gene ID: 98256, updated on 17-Aug-2019 Gene summary Official Symbol Kmo provided by MGI Official Full Name kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) provided by MGI Primary source MGI:MGI:2138151 See related Ensembl:ENSMUSG00000039783 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 AI046660 Expression Biased expression in liver adult (RPKM 28.0), kidney adult (RPKM 18.4) and 6 other tissuesS ee more Orthologs human all Genomic context Location: 1; 1 H3 See Kmo in Genome Data Viewer Exon count: 15 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (175632193..175660853) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (177562324..177590984) Chromosome 1 - NC_000067.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Kmo ENSMUSG00000039783 Description kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) [Source:MGI Symbol;Acc:MGI:2138151] Location Chromosome 1: 175,620,381-175,662,116 forward strand. GRCm38:CM000994.2 About this gene This gene has 6 transcripts (splice variants), 205 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Kmo- ENSMUST00000040250.14 2695 479aa ENSMUSP00000038914.8 Protein coding CCDS15548 Q91WN4 TSL:1 201 GENCODE basic APPRIS P2 Kmo- ENSMUST00000097458.3 3672 445aa ENSMUSP00000095067.3 Protein coding - Q91WN4 TSL:5 202 GENCODE basic APPRIS ALT2 Kmo- ENSMUST00000140474.7 1528 255aa ENSMUSP00000122943.1 Nonsense mediated - Q3V0K6 TSL:1 204 decay Kmo- ENSMUST00000142223.7 1600 No - Retained intron - - TSL:1 205 protein Kmo- ENSMUST00000150646.1 459 No - lncRNA - - TSL:3 206 protein Kmo- ENSMUST00000137174.1 374 No - lncRNA - - TSL:5 203 protein Page 6 of 8 https://www.alphaknockout.com 61.74 kb Forward strand 175.62Mb 175.63Mb 175.64Mb 175.65Mb 175.66Mb 175.67Mb Genes (Comprehensive set... Kmo-204 >nonsense mediated decay Kmo-201 >protein coding Kmo-205 >retained intron Kmo-206 >lncRNA Kmo-202 >protein coding Kmo-203 >lncRNA Contigs AC124366.4 > Genes < Fh1-201protein coding < Opn3-201protein coding (Comprehensive set... < Fh1-204nonsense mediated decay Regulatory Build 175.62Mb 175.63Mb 175.64Mb 175.65Mb 175.66Mb 175.67Mb Reverse strand 61.74 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 RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000040250 28.86 kb Forward strand Kmo-201 >protein coding ENSMUSP00000038... Transmembrane heli... Low complexity (Seg) Superfamily FAD/NAD(P)-binding domain superfamily Prints PR00420 Pfam FAD-binding domain PANTHER PTHR46028 PTHR46028:SF2 HAMAP Kynurenine 3-monooxygenase Gene3D FAD/NAD(P)-binding domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 479 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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