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Mouse Anapc1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Anapc1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Anapc1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Anapc1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Anapc1 gene (NCBI Reference Sequence: NM_008569 ; Ensembl: ENSMUSG00000014355 ) is located on Mouse chromosome 2. 48 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 48 (Transcript: ENSMUST00000014499). Exon 8~10 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Anapc1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-402F15 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: Exon 8 starts from about 11.76% of the coding region. The knockout of Exon 8~10 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 1963 bp, and the size of intron 10 for 3'-loxP site insertion: 2393 bp. The size of effective cKO region: ~2197 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 7 8 9 10 48 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Anapc1 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(8697bp) | A(26.34% 2291) | C(18.86% 1640) | T(33.49% 2913) | G(21.31% 1853) 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% chr2 - 128676498 128679497 3000 browser details YourSeq 72 2420 2541 3000 82.8% chr18 + 56593508 56593620 113 browser details YourSeq 69 2816 2944 3000 85.4% chr14 - 122242319 122242442 124 browser details YourSeq 68 2453 2844 3000 94.8% chr11 - 101803159 101803648 490 browser details YourSeq 68 2813 2897 3000 93.3% chr16 + 10986114 10986197 84 browser details YourSeq 67 2804 2925 3000 89.6% chr4 + 133785716 133785844 129 browser details YourSeq 63 2437 2920 3000 92.0% chr19 - 40638445 40638973 529 browser details YourSeq 63 2404 2496 3000 81.5% chr4 + 150399360 150399430 71 browser details YourSeq 61 2150 2318 3000 88.9% chr4 - 44470649 44470816 168 browser details YourSeq 59 2151 2302 3000 87.2% chr10 + 68242587 68242751 165 browser details YourSeq 59 2420 2503 3000 83.4% chr1 + 154069397 154069476 80 browser details YourSeq 59 2420 2503 3000 83.4% chr1 + 68736512 68736591 80 browser details YourSeq 58 2806 2871 3000 94.0% chr8 + 51029370 51029435 66 browser details YourSeq 58 2815 2897 3000 84.8% chr8 + 13056059 13056138 80 browser details YourSeq 57 2814 2897 3000 85.1% chr12 - 90287347 90287426 80 browser details YourSeq 56 2812 2877 3000 92.5% chr2 + 65771486 65771551 66 browser details YourSeq 55 1911 2230 3000 67.2% chr9 - 42954372 42954468 97 browser details YourSeq 55 2422 2501 3000 86.0% chr17 - 24871336 24871411 76 browser details YourSeq 55 2215 2381 3000 85.3% chr1 + 60367360 60367519 160 browser details YourSeq 54 2170 2444 3000 93.5% chr5 - 130290090 130290507 418 Note: The 3000 bp section upstream of Exon 8 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% chr2 - 128671301 128674300 3000 browser details YourSeq 190 306 881 3000 84.1% chr1 - 59836647 59837163 517 browser details YourSeq 182 318 878 3000 88.7% chr10 - 88471342 88837767 366426 browser details YourSeq 173 271 869 3000 87.4% chr19 - 4433404 4434124 721 browser details YourSeq 150 2519 2683 3000 95.8% chr4 - 87030349 87030529 181 browser details YourSeq 150 274 544 3000 84.5% chr15 - 58027007 58027312 306 browser details YourSeq 149 2516 2684 3000 94.1% chr3 - 95749194 95749362 169 browser details YourSeq 146 2508 2682 3000 93.0% chr11 + 74802542 74802718 177 browser details YourSeq 143 2541 2779 3000 86.8% chr10 - 79693337 79693496 160 browser details YourSeq 139 2517 2683 3000 93.3% chr13 - 8868126 8868301 176 browser details YourSeq 138 712 881 3000 90.6% chr16 - 60162342 60162511 170 browser details YourSeq 136 709 881 3000 91.5% chr9 + 64158076 64158252 177 browser details YourSeq 133 713 881 3000 95.3% chr7 - 112643490 112643659 170 browser details YourSeq 132 712 898 3000 91.8% chr5 + 31678827 31679008 182 browser details YourSeq 131 2541 2682 3000 96.5% chr12 - 79439295 79439437 143 browser details YourSeq 131 711 881 3000 88.9% chr6 + 30002106 30002278 173 browser details YourSeq 130 715 878 3000 90.3% chr2 - 119892472 119892638 167 browser details YourSeq 128 711 881 3000 87.6% chr3 - 88691353 88691524 172 browser details YourSeq 128 2543 2745 3000 86.4% chr10 - 62968536 62968674 139 browser details YourSeq 128 2543 2684 3000 95.1% chr2 + 34393498 34393639 142 Note: The 3000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Anapc1 anaphase promoting complex subunit 1 [ Mus musculus (house mouse) ] Gene ID: 17222, updated on 10-Oct-2019 Gene summary Official Symbol Anapc1 provided by MGI Official Full Name anaphase promoting complex subunit 1 provided by MGI Primary source MGI:MGI:103097 See related Ensembl:ENSMUSG00000014355 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 Apc1; Mcpr; tsg24; AI047775; AI853536; AW547281; 2610021O03Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 13.6), limb E14.5 (RPKM 12.9) and 28 other tissues See more Orthologs human all Genomic context Location: 2; 2 F1 See Anapc1 in Genome Data Viewer Exon count: 50 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (128610083..128687406, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (128435819..128513131, complement) Chromosome 2 - NC_000068.7 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 11 transcripts Gene: Anapc1 ENSMUSG00000014355 Description anaphase promoting complex subunit 1 [Source:MGI Symbol;Acc:MGI:103097] Gene Synonyms 2610021O03Rik, Apc1, Mcpr, tsg24 Location Chromosome 2: 128,610,104-128,687,391 reverse strand. GRCm38:CM000995.2 About this gene This gene has 11 transcripts (splice variants), 216 orthologues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Anapc1-201 ENSMUST00000014499.9 8942 1944aa ENSMUSP00000014499.3 Protein coding CCDS16715 P53995 TSL:1 GENCODE basic APPRIS P1 Anapc1-203 ENSMUST00000110333.1 4214 1213aa ENSMUSP00000105962.1 Protein coding - A2ATQ5 TSL:1 GENCODE basic Anapc1-202 ENSMUST00000110332.1 1214 80aa ENSMUSP00000105961.1 Protein coding - Q6PG65 TSL:1 GENCODE basic Anapc1-204 ENSMUST00000123503.1 3261 No protein - lncRNA - - TSL:1 Anapc1-206 ENSMUST00000134485.1 3007 No protein - lncRNA - - TSL:1 Anapc1-209 ENSMUST00000151171.1 836 No protein - lncRNA - - TSL:2 Anapc1-208 ENSMUST00000145906.7 742 No protein - lncRNA - - TSL:2 Anapc1-205 ENSMUST00000124418.1 691 No protein - lncRNA - - TSL:2 Anapc1-210 ENSMUST00000154320.1 667 No protein - lncRNA - - TSL:3 Anapc1-207 ENSMUST00000143007.1 627 No protein - lncRNA - - TSL:3 Anapc1-211 ENSMUST00000154995.1 564 No protein - lncRNA - - TSL:3 Page 6 of 8 https://www.alphaknockout.com 97.29 kb Forward strand Genes Gm14007-201 >processed pseudogene (Comprehensive set... Contigs AL928910.7 > Genes (Comprehensive set... < Gm39929-201lncRNA < Anapc1-205lncRNA < Anapc1-210lncRNA < Anapc1-203protein coding < Anapc1-201protein coding < Anapc1-209lncRNA < Anapc1-204lncRNA < Anapc1-206lncRNA < Anapc1-208lncRNA < Anapc1-207lncRNA < Anapc1-202protein coding < Anapc1-211lncRNA Regulatory Build Reverse strand 97.29 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000014499 < Anapc1-201protein coding Reverse strand 77.29 kb ENSMUSP00000014... MobiDB lite Low complexity (Seg) Pfam Anaphase-promoting complex subunit 1 Anaphase-promoting complex subunit 1, C-terminal Proteasome/cyclosome repeat PANTHER Anaphase-promoting complex subunit 1 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 200 400 600 800 1000 1200 1400 1600 1944 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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