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Mouse Acad8 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Acad8 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Acad8 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Acad8 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Acad8 gene (NCBI Reference Sequence: NM_025862 ; Ensembl: ENSMUSG00000031969 ) is located on Mouse chromosome 9. 11 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 11 (Transcript: ENSMUST00000060513). 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 Acad8 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-41F5 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 an ENU-induced allele exhibit cold intolerance at young age with a progressive hepatic steatosis and abnormal mitochondria. Exon 2 starts from about 8.39% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 7133 bp, and the size of intron 2 for 3'-loxP site insertion: 750 bp. The size of effective cKO region: ~601 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 3 4 5 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Acad8 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(7101bp) | A(25.45% 1807) | C(22.5% 1598) | T(31.32% 2224) | G(20.73% 1472) 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 - 26992511 26995510 3000 browser details YourSeq 68 576 1057 3000 70.9% chr12 + 38713565 38713738 174 browser details YourSeq 65 571 1056 3000 67.9% chr1 - 153403701 153403861 161 browser details YourSeq 55 2639 2728 3000 92.5% chr1 + 189753883 189754387 505 browser details YourSeq 52 953 1053 3000 86.2% chr13 + 95613635 95613916 282 browser details YourSeq 51 2600 2671 3000 80.8% chr18 + 6754905 6754962 58 browser details YourSeq 49 977 1053 3000 79.1% chr4 - 126307908 126307976 69 browser details YourSeq 49 2566 2652 3000 90.2% chr1 - 80256406 80256559 154 browser details YourSeq 45 577 978 3000 56.7% chr2 - 180138049 180138108 60 browser details YourSeq 42 2600 2676 3000 95.6% chr4 + 17722830 17722907 78 browser details YourSeq 40 130 234 3000 89.2% chr14 - 11976450 11976553 104 browser details YourSeq 39 957 1015 3000 83.1% chr12 - 59215988 59216046 59 browser details YourSeq 38 105 224 3000 79.7% chr10 - 18334493 18334610 118 browser details YourSeq 37 955 1013 3000 83.7% chr12 + 59973396 59973455 60 browser details YourSeq 34 118 179 3000 94.9% chr10 - 24717680 24717742 63 browser details YourSeq 34 103 140 3000 94.8% chr6 + 113348231 113348268 38 browser details YourSeq 33 120 175 3000 94.5% chr12 + 55127172 55127227 56 browser details YourSeq 33 120 175 3000 94.5% chr12 + 55202236 55202291 56 browser details YourSeq 32 971 1015 3000 90.0% chr8 - 107414746 107414792 47 browser details YourSeq 32 974 1014 3000 90.0% chr13 + 109206916 109206958 43 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% chr9 - 26988910 26991909 3000 browser details YourSeq 273 2111 2833 3000 82.4% chr13 - 42600546 42601458 913 browser details YourSeq 195 57 2991 3000 91.9% chr5 - 147834224 147994756 160533 browser details YourSeq 180 2068 2758 3000 85.8% chr4 + 69065121 69065817 697 browser details YourSeq 172 2278 2790 3000 81.0% chr11 - 56175163 56175806 644 browser details YourSeq 167 2071 2521 3000 81.3% chr8 - 96143613 96144066 454 browser details YourSeq 163 2191 2831 3000 83.5% chr10 - 130273584 130274403 820 browser details YourSeq 161 26 2375 3000 88.6% chr11 + 63676756 64076156 399401 browser details YourSeq 158 1956 2414 3000 87.8% chr6 + 128623078 128623538 461 browser details YourSeq 149 2135 2523 3000 91.3% chr12 - 54914629 54915194 566 browser details YourSeq 148 2146 2600 3000 86.3% chr5 + 20305877 20306454 578 browser details YourSeq 145 2135 2523 3000 84.7% chr4 + 100206083 100206482 400 browser details YourSeq 142 2247 2678 3000 79.9% chr13 - 90056067 90056564 498 browser details YourSeq 135 2136 2623 3000 84.5% chr14 - 36636078 36636751 674 browser details YourSeq 132 2042 2525 3000 84.2% chr15 + 40762684 40763162 479 browser details YourSeq 132 1996 2348 3000 89.8% chr11 + 70413620 70413974 355 browser details YourSeq 129 2109 2461 3000 73.9% chrX - 60336807 60337216 410 browser details YourSeq 128 1940 2378 3000 79.2% chr7 + 125791308 125791701 394 browser details YourSeq 124 2198 2712 3000 81.7% chr4 - 144368235 144368806 572 browser details YourSeq 120 2142 2375 3000 88.2% chr15 + 6944665 6944893 229 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: Acad8 acyl-Coenzyme A dehydrogenase family, member 8 [ Mus musculus (house mouse) ] Gene ID: 66948, updated on 12-Aug-2019 Gene summary Official Symbol Acad8 provided by MGI Official Full Name acyl-Coenzyme A dehydrogenase family, member 8 provided by MGI Primary source MGI:MGI:1914198 See related Ensembl:ENSMUSG00000031969 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 AI786953; 2310016C19Rik Expression Ubiquitous expression in kidney adult (RPKM 8.3), genital fat pad adult (RPKM 7.1) and 28 other tissues See more Orthologs human all Genomic context Location: 9; 9 A4 See Acad8 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (26974138..26999571, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (26781723..26807134, complement) Chromosome 9 - NC_000075.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Acad8 ENSMUSG00000031969 Description acyl-Coenzyme A dehydrogenase family, member 8 [Source:MGI Symbol;Acc:MGI:1914198] Gene Synonyms 2310016C19Rik Location Chromosome 9: 26,974,135-26,999,566 reverse strand. GRCm38:CM001002.2 About this gene This gene has 8 transcripts (splice variants), 195 orthologues, 15 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Acad8- ENSMUST00000060513.7 2859 413aa ENSMUSP00000054370.7 Protein coding CCDS40567 A0A0R4J0P1 TSL:1 201 GENCODE basic APPRIS P2 Acad8- ENSMUST00000120367.7 2885 413aa ENSMUSP00000112908.1 Protein coding - D3YTT4 TSL:5 202 GENCODE basic APPRIS ALT2 Acad8- ENSMUST00000128923.7 852 49aa ENSMUSP00000122444.1 Nonsense mediated - D6RDD5 TSL:5 203 decay Acad8- ENSMUST00000132293.1 679 49aa ENSMUSP00000123012.1 Nonsense mediated - D6RDD5 TSL:3 205 decay Acad8- ENSMUST00000215693.1 661 66aa ENSMUSP00000150728.1 Nonsense mediated - A0A1L1SUG2 CDS 5' 208 decay incomplete TSL:5 Acad8- ENSMUST00000129490.7 3188 No - Retained intron - - TSL:5 204 protein Acad8- ENSMUST00000151075.1 785 No - Retained intron - - TSL:1 207 protein Acad8- ENSMUST00000138102.1 600 No - Retained intron - - TSL:2 206 protein Page 6 of 8 https://www.alphaknockout.com 45.43 kb Forward strand 26.97Mb 26.98Mb 26.99Mb 27.00Mb Genes Thyn1-203 >protein coding (Comprehensive set... Thyn1-205 >retained intron Thyn1-202 >protein coding Thyn1-201 >protein coding Thyn1-204 >retained intron Thyn1-208 >retained intron Thyn1-206 >retained intron Thyn1-207 >retained intron Contigs AC138616.4 > < AC156163.2 Genes (Comprehensive set... < Acad8-202protein coding < Vps26b-201protein coding < Acad8-204retained intron < Acad8-201protein coding < Acad8-207retained intron < Acad8-206retained intron < Acad8-208nonsense mediated decay < Acad8-203nonsense mediated decay < Acad8-205nonsense mediated decay Regulatory Build 26.97Mb 26.98Mb 26.99Mb 27.00Mb Reverse strand 45.43 kb Regulation Legend Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000060513 < Acad8-201protein coding Reverse strand 25.41 kb ENSMUSP00000054... Superfamily Acyl-CoA dehydrogenase/oxidase, N-terminal and middle domain superfamily Acyl-CoA dehydrogenase-like, C-terminal Pfam Acyl-CoA dehydrogenase/oxidase, N-terminal Acyl-CoA dehydrogenase/oxidase C-terminal Acyl-CoA oxidase/dehydrogenase, central domain PROSITE patterns Acyl-CoA dehydrogenase, conserved site PIRSF PIRSF016578 PANTHER PTHR43831 PTHR43831:SF1 Gene3D Acyl-CoA dehydrogenase/oxidase, N-terminal domain superfamily 1.20.140.10 2.40.110.10 CDD Isobutyryl-CoA dehydrogenase All sequence SNPs/i..
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