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Mouse Mcoln3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Mcoln3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Mcoln3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Mcoln3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mcoln3 gene (NCBI Reference Sequence: NM_134160 ; Ensembl: ENSMUSG00000036853 ) is located on Mouse chromosome 3. 12 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 12 (Transcript: ENSMUST00000039450). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Mcoln3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-90O23 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: Heterozygotes show normal/diluted/white hair patches, circling, hyperactivity, deafness, and reduced fertility. Homozygotes are white with small patches of color and show severe behavioral abnormalities, poor postnatal viability and are nearly infertile. Exon 6 starts from about 44.18% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 1954 bp, and the size of intron 6 for 3'-loxP site insertion: 2403 bp. The size of effective cKO region: ~600 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 4 5 6 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Mcoln3 Homology arm cKO region 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(7100bp) | A(26.25% 1864) | C(23.01% 1634) | T(28.69% 2037) | G(22.04% 1565) 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% chr3 + 146127898 146130897 3000 browser details YourSeq 153 544 738 3000 89.3% chr14 - 34394814 34394983 170 browser details YourSeq 147 528 703 3000 91.1% chr17 - 71340340 71340511 172 browser details YourSeq 146 542 712 3000 94.0% chr12 + 109714371 109714546 176 browser details YourSeq 145 536 713 3000 92.0% chr14 + 40770353 40770531 179 browser details YourSeq 145 544 707 3000 95.7% chr11 + 29699082 29699256 175 browser details YourSeq 143 542 737 3000 85.3% chr11 + 14687815 14687985 171 browser details YourSeq 142 544 738 3000 87.0% chr15 - 81681588 81681749 162 browser details YourSeq 142 536 709 3000 91.4% chr7 + 101864966 101865134 169 browser details YourSeq 141 542 711 3000 88.9% chr4 - 108169220 108169381 162 browser details YourSeq 141 556 745 3000 89.2% chr1 + 13665815 13665984 170 browser details YourSeq 140 544 713 3000 90.2% chr15 + 77341846 77342010 165 browser details YourSeq 140 542 711 3000 89.4% chr1 + 55189432 55189593 162 browser details YourSeq 139 545 738 3000 85.7% chrX - 73761388 73761558 171 browser details YourSeq 138 544 738 3000 86.5% chrY - 1217942 1218108 167 browser details YourSeq 138 544 713 3000 91.7% chr1 - 11127227 11127393 167 browser details YourSeq 137 542 712 3000 88.9% chr11 - 76181074 76181225 152 browser details YourSeq 137 544 708 3000 93.6% chr10 - 33434580 33434783 204 browser details YourSeq 137 544 709 3000 90.0% chr1 - 60210873 60211033 161 browser details YourSeq 137 556 710 3000 94.9% chr4 + 11312805 11312964 160 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% chr3 + 146131498 146134497 3000 browser details YourSeq 91 2688 2917 3000 97.0% chr2 + 143681668 143681946 279 browser details YourSeq 77 2594 2858 3000 87.3% chr15 - 102860999 102861397 399 browser details YourSeq 47 2806 2860 3000 94.5% chr2 + 44877600 44877662 63 browser details YourSeq 43 2726 2779 3000 87.8% chr10 + 73003435 73003486 52 browser details YourSeq 43 2639 2750 3000 72.0% chr1 + 156210327 156210405 79 browser details YourSeq 42 898 1037 3000 77.1% chr11 - 23773325 23773452 128 browser details YourSeq 42 2818 2871 3000 88.9% chr1 - 165747490 165747543 54 browser details YourSeq 41 2811 2868 3000 86.3% chr6 + 82842386 82842444 59 browser details YourSeq 41 899 1000 3000 68.9% chr2 + 134583625 134583678 54 browser details YourSeq 39 2806 2851 3000 93.5% chr17 - 57079616 57079663 48 browser details YourSeq 38 1008 1081 3000 95.3% chr16 + 10248500 10248574 75 browser details YourSeq 38 1056 1109 3000 79.2% chr14 + 57761834 57761882 49 browser details YourSeq 38 2818 2873 3000 85.5% chr1 + 80204713 80204767 55 browser details YourSeq 37 2806 2857 3000 86.6% chr1 + 62880108 62880161 54 browser details YourSeq 36 2818 2861 3000 91.0% chr14 - 67140695 67140738 44 browser details YourSeq 35 2806 2863 3000 92.9% chr11 + 50141578 50141639 62 browser details YourSeq 34 2815 2850 3000 97.3% chr13 - 57093156 57093191 36 browser details YourSeq 33 1056 1108 3000 74.5% chr10 - 7687946 7687993 48 browser details YourSeq 33 1008 1081 3000 84.3% chr17 + 36018700 36018771 72 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Mcoln3 mucolipin 3 [ Mus musculus (house mouse) ] Gene ID: 171166, updated on 12-Oct-2019 Gene summary Official Symbol Mcoln3 provided by MGI Official Full Name mucolipin 3 provided by MGI Primary source MGI:MGI:1890500 See related Ensembl:ENSMUSG00000036853 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 Va; TRPML3; 6720490O21Rik Expression Biased expression in placenta adult (RPKM 3.2), kidney adult (RPKM 2.7) and 9 other tissues See more Orthologs human all Genomic context Location: 3 H2; 3 71.03 cM See Mcoln3 in Genome Data Viewer Exon count: 15 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (146093277..146141808) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (145784755..145803610) Chromosome 3 - NC_000069.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Mcoln3 ENSMUSG00000036853 Description mucolipin 3 [Source:MGI Symbol;Acc:MGI:1890500] Gene Synonyms 6720490O21Rik, TRPML3, Va, varitint-waddler Location Chromosome 3: 146,117,450-146,141,806 forward strand. GRCm38:CM000996.2 About this gene This gene has 3 transcripts (splice variants), 256 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 24 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Mcoln3- ENSMUST00000039450.4 2991 553aa ENSMUSP00000038801.4 Protein coding CCDS17900 Q3KP78 TSL:1 201 Q8R4F0 GENCODE basic APPRIS P1 Mcoln3- ENSMUST00000140214.2 477 96aa ENSMUSP00000115655.1 Protein coding - D3YZE2 CDS 3' 202 incomplete TSL:2 Mcoln3- ENSMUST00000146689.2 698 No - Retained - - TSL:2 203 protein intron 44.36 kb Forward strand 146.11Mb 146.12Mb 146.13Mb 146.14Mb 146.15Mb Genes (Comprehensive set... Mcoln3-202 >protein coding Mcoln3-203 >retained intron Mcoln2-203 >retained intron Mcoln3-201 >protein coding Mcoln2-201 >protein coding Mcoln2-202 >protein coding Contigs AC161212.9 > Genes < Wdr63-201protein coding < Gm25627-201snRNA (Comprehensive set... Regulatory Build 146.11Mb 146.12Mb 146.13Mb 146.14Mb 146.15Mb Reverse strand 44.36 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000039450 20.14 kb Forward strand Mcoln3-201 >protein coding ENSMUSP00000038... Transmembrane heli... Low complexity (Seg) Pfam Polycystin cation channel, PKD1/PKD2 PANTHER PTHR12127:SF5 Mucolipin All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 553 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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