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Mouse Myl6 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Myl6 Conditional Knockout Project (CRISPR/Cas9) http://www.alphaknockout.com/ Mouse Myl6 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Myl6 conditional knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Myl6 gene (NCBI Reference Sequence: NM_010860 ; Ensembl: ENSMUSG00000090841 ) is located on Mouse chromosome 10. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000164181). Exon 3~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Myl6 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-128E20 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: The expression of mouse Smarcc2 may be affected by deletion of this cKO region. Exon 3~6 is not frameshift exon, and covers 93.16% of the coding region. The size of intron 2 for 5'-loxP site insertion: 793 bp. The size of effective cKO region: ~2636 bp. The cKO region does not have any other known gene. Page 1 of 8 http://www.alphaknockout.com/ Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 6 7 1 2 3 4 5 6 28 27 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Myl6b Exon of mouse Myl6 cKO region Exon of mouse Smarcc2 loxP site Page 2 of 8 http://www.alphaknockout.com/ Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement 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 Summary: Full Length(8386bp) | A(22.72% 1905) | C(24.3% 2038) | G(28.01% 2349) | T(24.97% 2094) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. Page 3 of 8 http://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% chr10 - 128492996 128495995 3000 browser details YourSeq 117 789 1404 3000 72.1% chr11 + 104584459 104585012 554 browser details YourSeq 84 2121 2457 3000 76.6% chr9 - 44375166 44375259 94 browser details YourSeq 84 792 925 3000 81.4% chr9 + 110767931 110768064 134 browser details YourSeq 79 789 913 3000 81.6% chr14 - 55420405 55420529 125 browser details YourSeq 77 789 913 3000 80.8% chr8 - 105660470 105660594 125 browser details YourSeq 75 75 341 3000 73.2% chr2 + 91225580 91225691 112 browser details YourSeq 68 2145 2457 3000 74.7% chr1 + 45857741 45857811 71 browser details YourSeq 66 184 341 3000 75.9% chr18 + 53980742 53980861 120 browser details YourSeq 65 2142 2453 3000 72.9% chr4 + 14433848 14433917 70 browser details YourSeq 64 789 886 3000 82.7% chr1 - 45857495 45857592 98 browser details YourSeq 64 243 337 3000 89.2% chr13 + 29229997 29230094 98 browser details YourSeq 62 249 352 3000 87.1% chr18 + 19454539 19454645 107 browser details YourSeq 61 243 341 3000 88.8% chr3 - 43390019 43390120 102 browser details YourSeq 60 75 341 3000 71.3% chr3 - 45594284 45594395 112 browser details YourSeq 59 2121 2187 3000 94.1% chr14 - 55420722 55420788 67 browser details YourSeq 58 75 313 3000 68.9% chr1 - 34484283 34484364 82 browser details YourSeq 58 62 302 3000 67.2% chr2 + 152875001 152875076 76 browser details YourSeq 58 75 309 3000 69.7% chr1 + 36427003 36427153 151 browser details YourSeq 57 76 341 3000 69.5% chr10 - 93072312 93072423 112 Note: The 3000 bp section upstream of Exon 3 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% chr10 - 128487860 128490859 3000 browser details YourSeq 38 494 538 3000 82.1% chr11 + 63675954 63675992 39 browser details YourSeq 36 494 538 3000 79.5% chr11 + 63676320 63676358 39 browser details YourSeq 29 1483 1572 3000 96.8% chr15 - 71217264 71217354 91 browser details YourSeq 26 491 518 3000 96.5% chr15 - 101019129 101019156 28 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 http://www.alphaknockout.com/ Gene and protein information: Myl6 myosin, light polypeptide 6, alkali, smooth muscle and non-muscle [ Mus musculus (house mouse) ] Gene ID: 17904, updated on 24-Oct-2019 Gene summary Official Symbol Myl6 provided by MGI Official Full Name myosin, light polypeptide 6, alkali, smooth muscle and non-muscle provided by MGI Primary source MGI:MGI:109318 See related Ensembl:ENSMUSG00000090841 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 LC17; Myln; ESMLC; MLC-3; MLC1SM; LC17-GI Expression Broad expression in bladder adult (RPKM 2188.3), placenta adult (RPKM 580.4) and 23 other tissues See more Orthologs human all Genomic context Location: 10; 10 D3 See Myl6 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (128490859..128493886, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (127927917..127930881, complement) Chromosome 10 - NC_000076.6 Page 5 of 8 http://www.alphaknockout.com/ Transcript information: This gene has 15 transcripts Gene: Myl6 ENSMUSG00000090841 Description myosin, light polypeptide 6, alkali, smooth muscle and non-muscle [Source:MGI Symbol;Acc:MGI:109318] Gene Synonyms MLC3nm, Myln Location Chromosome 10: 128,490,860-128,494,145 reverse strand. GRCm38:CM001003.2 About this gene This gene has 15 transcripts (splice variants), 299 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Myl6- ENSMUST00000164181.1 665 151aa ENSMUSP00000128803.1 Protein coding CCDS48728 Q60605 TSL:1 201 Q642K0 GENCODE basic APPRIS P2 Myl6- ENSMUST00000217733.1 1668 152aa ENSMUSP00000151236.1 Protein coding - A0A1W2P6F6 TSL:1 202 GENCODE basic Myl6- ENSMUST00000219236.1 692 158aa ENSMUSP00000151915.1 Protein coding - A0A1W2P7Q9 TSL:2 211 GENCODE basic Myl6- ENSMUST00000218127.1 691 151aa ENSMUSP00000151693.1 Protein coding - Q60605 TSL:2 206 GENCODE basic APPRIS ALT1 Myl6- ENSMUST00000217969.1 608 139aa ENSMUSP00000151250.1 Protein coding - A0A1W2P6G5 TSL:2 205 GENCODE basic Myl6- ENSMUST00000217776.1 562 158aa ENSMUSP00000151786.1 Protein coding - A0A1W2P7Q9 TSL:3 203 GENCODE basic Myl6- ENSMUST00000220427.1 746 38aa ENSMUSP00000151914.1 Nonsense mediated - A0A1W2P888 TSL:3 215 decay Myl6- ENSMUST00000220307.1 496 46aa ENSMUSP00000151991.1 Nonsense mediated - A0A1W2P8F0 TSL:5 214 decay Myl6- ENSMUST00000218813.1 3040 No - Retained intron - - TSL:NA 209 protein Myl6- ENSMUST00000218170.1 824 No - Retained intron - - TSL:2 207 protein Myl6- ENSMUST00000219554.1 790 No - Retained intron - - TSL:2 212 protein Myl6- ENSMUST00000217913.1 436 No - Retained intron - - TSL:1 204 protein Myl6- ENSMUST00000219655.1 651 No - lncRNA - - TSL:2 213 protein Myl6- ENSMUST00000219100.1 620 No - lncRNA - - TSL:3 210 protein Myl6- ENSMUST00000218713.1 369 No - lncRNA - - TSL:5 208 protein Page 6 of 8 http://www.alphaknockout.com/ 23.29 kb Forward strand 128.485Mb 128.490Mb 128.495Mb 128.500Mb Genes Smarcc2-205 >protein coding (Comprehensive set... Smarcc2-201 >protein coding Smarcc2-202 >protein coding Smarcc2-203 >protein coding Smarcc2-204 >retained intron Contigs AC170752.2 > Genes (Comprehensive set... < Myl6-201protein coding < Myl6b-201protein coding < Myl6-209retained intron < Myl6b-202retained intron < Myl6-206protein coding < Myl6-202protein coding < Myl6-212retained intron < Myl6-213lncRNA < Myl6-205protein coding < Myl6-211protein coding < Myl6-203protein coding < Myl6-208lncRNA < Myl6-214nonsense mediated decay < Myl6-215nonsense mediated decay < Myl6-207retained intron < Myl6-210lncRNA < Myl6-204retained intron Regulatory Build 128.485Mb 128.490Mb 128.495Mb 128.500Mb Reverse strand 23.29 kb Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Page 7 of 8 http://www.alphaknockout.com/ Transcript: ENSMUST00000164181 < Myl6-201protein coding Reverse strand 3.02 kb ENSMUSP00000128... Superfamily EF-hand domain pair SMART EF-hand domain Pfam EF-hand domain PROSITE profiles EF-hand domain PANTHER PTHR23048 PTHR23048:SF7 Gene3D 1.10.238.10 CDD EF-hand domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 151 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC, VectorBuilder. Page 8 of 8.
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