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Mouse Ift20 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ift20 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ift20 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ift20 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ift20 gene (NCBI Reference Sequence: NM_018854 ; Ensembl: ENSMUSG00000001105 ) is located on Mouse chromosome 11. 6 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000128788). 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 Ift20 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-356D12 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 null mutation die before birth. Mice with conditional loss in renal collecting ducts lackprimary cilia and develop renal cysts. Exon 4 starts from about 32.32% 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: 997 bp, and the size of intron 4 for 3'-loxP site insertion: 849 bp. The size of effective cKO region: ~586 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 4 5 6 3 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ift20 Homology arm cKO region Exon of mouse Tmem97 loxP site Page 2 of 7 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(7086bp) | A(24.74% 1753) | C(23.14% 1640) | T(27.9% 1977) | G(24.22% 1716) 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 7 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% chr11 + 78536711 78539710 3000 browser details YourSeq 143 698 859 3000 92.1% chr16 + 8799447 8799597 151 browser details YourSeq 140 717 869 3000 96.7% chrX - 60636654 60636811 158 browser details YourSeq 138 721 868 3000 97.3% chrX + 7254449 7254609 161 browser details YourSeq 137 713 867 3000 92.8% chr15 - 100252259 100252411 153 browser details YourSeq 137 717 868 3000 95.4% chr15 + 82635801 82635952 152 browser details YourSeq 137 717 868 3000 95.4% chr14 + 10153615 10153768 154 browser details YourSeq 137 697 868 3000 91.2% chr11 + 99004862 99005031 170 browser details YourSeq 136 717 868 3000 93.4% chr12 - 77259971 77260121 151 browser details YourSeq 136 717 868 3000 93.3% chr17 + 73807965 73808114 150 browser details YourSeq 136 726 878 3000 96.6% chr11 + 89004988 89005598 611 browser details YourSeq 135 717 869 3000 93.4% chr5 + 136178871 136179022 152 browser details YourSeq 135 721 868 3000 96.0% chr5 + 121391009 121391157 149 browser details YourSeq 135 717 868 3000 94.8% chr2 + 59500951 59501102 152 browser details YourSeq 134 714 868 3000 94.2% chr3 + 57647579 57647736 158 browser details YourSeq 133 717 868 3000 95.3% chr4 - 149291874 149292397 524 browser details YourSeq 133 721 869 3000 93.3% chr4 - 136432494 136432641 148 browser details YourSeq 133 716 863 3000 96.6% chrX + 103418540 103418687 148 browser details YourSeq 132 717 867 3000 96.6% chrX + 142419216 142419366 151 browser details YourSeq 132 725 867 3000 97.2% chr4 + 118352730 118352872 143 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% chr11 + 78540297 78543296 3000 browser details YourSeq 956 1521 2502 3000 99.0% chr16 - 22230660 22231641 982 browser details YourSeq 276 597 1418 3000 86.5% chr9 + 12234192 12234705 514 browser details YourSeq 91 193 2926 3000 88.2% chr1 - 9711930 9929823 217894 browser details YourSeq 75 156 286 3000 85.6% chr11 + 5782998 5783128 131 browser details YourSeq 72 148 263 3000 90.0% chr2 - 144212681 144212797 117 browser details YourSeq 72 153 263 3000 86.0% chr1 + 186545225 186545336 112 browser details YourSeq 70 125 282 3000 84.3% chr4 - 117533823 117533975 153 browser details YourSeq 68 2847 2953 3000 94.9% chr5 - 65332279 65332393 115 browser details YourSeq 68 2864 2953 3000 93.6% chr6 + 83528541 83528631 91 browser details YourSeq 67 2864 2965 3000 88.0% chr8 + 25549909 25550011 103 browser details YourSeq 65 149 267 3000 91.2% chr5 - 100216022 100216141 120 browser details YourSeq 65 149 264 3000 88.3% chr10 + 60021350 60021466 117 browser details YourSeq 64 2827 2934 3000 93.5% chr11 - 116252545 116253103 559 browser details YourSeq 63 2864 2965 3000 85.6% chr11 - 76161960 76580859 418900 browser details YourSeq 63 120 240 3000 85.6% chr4 + 148390410 148390527 118 browser details YourSeq 62 137 290 3000 93.2% chr2 + 167550316 167550472 157 browser details YourSeq 62 107 217 3000 90.5% chr2 + 27060665 27060774 110 browser details YourSeq 62 2864 2964 3000 92.0% chr12 + 4100568 4100668 101 browser details YourSeq 61 109 287 3000 93.1% chr6 - 48710916 48711096 181 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ift20 intraflagellar transport 20 [ Mus musculus (house mouse) ] Gene ID: 55978, updated on 14-Oct-2019 Gene summary Official Symbol Ift20 provided by MGI Official Full Name intraflagellar transport 20 provided by MGI Primary source MGI:MGI:1915585 See related Ensembl:ENSMUSG00000001105 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 mIFT20; AU015496; 0610009H04Rik Expression Ubiquitous expression in duodenum adult (RPKM 42.4), small intestine adult (RPKM 37.8) and 28 other tissues See more Orthologs human all Genomic context Location: 11; 11 B5 See Ift20 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (78536361..78541732) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (78349938..78354975) Chromosome 11 - NC_000077.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Ift20 ENSMUSG00000001105 Description intraflagellar transport 20 [Source:MGI Symbol;Acc:MGI:1915585] Gene Synonyms 0610009H04Rik Location Chromosome 11: 78,536,361-78,541,737 forward strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 211 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 Ift20-203 ENSMUST00000128788.7 1141 132aa ENSMUSP00000118015.1 Protein coding CCDS25111 Q61025 TSL:1 GENCODE basic APPRIS P1 Ift20-202 ENSMUST00000108275.1 582 132aa ENSMUSP00000103910.1 Protein coding CCDS25111 Q61025 TSL:1 GENCODE basic APPRIS P1 Ift20-201 ENSMUST00000050366.14 735 111aa ENSMUSP00000051699.8 Protein coding - Z4YJL9 CDS 3' incomplete TSL:5 Ift20-204 ENSMUST00000137120.7 1167 No protein - Retained intron - - TSL:1 Ift20-205 ENSMUST00000152016.1 909 No protein - Retained intron - - TSL:2 25.38 kb Forward strand 78.53Mb 78.54Mb 78.55Mb Genes (Comprehensive set... Ift20-203 >protein coding Ift20-201 >protein coding Ift20-202 >protein coding Ift20-204 >retained intron Ift20-205 >retained intron Contigs AL591177.14 > Genes < Tnfaip1-202protein coding < Tmem97-201protein coding (Comprehensive set... < Tnfaip1-201protein coding Regulatory Build 78.53Mb 78.54Mb 78.55Mb Reverse strand 25.38 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000128788 5.38 kb Forward strand Ift20-203 >protein coding ENSMUSP00000118... Low complexity (Seg) Coiled-coils (Ncoils) Pfam Intraflagellar transport protein 20 PANTHER Intraflagellar transport protein 20 Scale bar 0 20 40 60 80 100 132 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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