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Mouse Med13 Knockout Project (CRISPR/Cas9)

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Mouse Med13 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Med13 Knockout Project (CRISPR/Cas9) Objective: To create a Med13 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Med13 gene (NCBI Reference Sequence: NM_001080931 ; Ensembl: ENSMUSG00000034297 ) is located on Mouse chromosome 11. 30 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 30 (Transcript: ENSMUST00000043624). Exon 2~3 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a conditional allele exhibited in the heart exhibit increased susceptibility to obesity and worsened glucose intolerance when fed a high fat diet. Exon 2 starts from about 1.03% of the coding region. Exon 2~3 covers 6.2% of the coding region. The size of effective KO region: ~9649 bp. The KO 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 2 3 30 Legends Exon of mouse Med13 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1951 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 3 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(1951bp) | A(22.81% 445) | C(19.94% 389) | T(35.32% 689) | G(21.94% 428) Note: The 1951 bp section upstream of Exon 2 is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(23.5% 470) | C(17.75% 355) | T(38.15% 763) | G(20.6% 412) Note: The 2000 bp section downstream of Exon 3 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 1951 1 1951 1951 100.0% chr11 - 86355509 86357459 1951 browser details YourSeq 31 1835 1876 1951 94.2% chr5 + 123667427 123667469 43 browser details YourSeq 30 1408 1438 1951 100.0% chr9 + 66388946 66388984 39 browser details YourSeq 29 1832 1872 1951 96.8% chr15 + 48069169 48069213 45 browser details YourSeq 29 1729 1761 1951 90.4% chr13 + 112264211 112264242 32 browser details YourSeq 28 1833 1861 1951 100.0% chr9 + 99398123 99398152 30 browser details YourSeq 27 1831 1858 1951 100.0% chr8 - 24309978 24310006 29 browser details YourSeq 27 1834 1861 1951 100.0% chr4 + 140057721 140057754 34 browser details YourSeq 26 1833 1859 1951 100.0% chr1 + 62338226 62338254 29 browser details YourSeq 26 1834 1860 1951 100.0% chr1 + 12207674 12207701 28 browser details YourSeq 24 1730 1753 1951 100.0% chr2 - 85066809 85066832 24 browser details YourSeq 24 1832 1858 1951 84.0% chr5 + 141342249 141342273 25 browser details YourSeq 24 1854 1877 1951 100.0% chr18 + 89633123 89633146 24 browser details YourSeq 23 333 358 1951 96.0% chr18 - 82376531 82376556 26 browser details YourSeq 22 1924 1945 1951 100.0% chr2 - 64809370 64809391 22 browser details YourSeq 22 1408 1429 1951 100.0% chr11 + 77158436 77158457 22 browser details YourSeq 21 1730 1750 1951 100.0% chr8 - 102598886 102598906 21 browser details YourSeq 21 1732 1752 1951 100.0% chr16 + 48191246 48191266 21 browser details YourSeq 21 1426 1446 1951 100.0% chr1 + 69376206 69376226 21 Note: The 1951 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 2000 1 2000 2000 100.0% chr11 - 86343860 86345859 2000 browser details YourSeq 130 363 764 2000 74.9% chr14 + 27701098 27701397 300 browser details YourSeq 119 316 827 2000 75.5% chr14 + 22120263 22120539 277 browser details YourSeq 101 167 527 2000 82.3% chr9 + 113736632 113737119 488 browser details YourSeq 100 338 533 2000 79.2% chr5 - 147647467 147647646 180 browser details YourSeq 98 324 533 2000 82.3% chr19 + 53182763 53182987 225 browser details YourSeq 95 441 941 2000 88.7% chr8 + 121456550 121457059 510 browser details YourSeq 92 396 535 2000 83.5% chr9 - 43029213 43029618 406 browser details YourSeq 92 398 536 2000 83.5% chr5 - 127753649 127753789 141 browser details YourSeq 88 330 532 2000 73.1% chr5 - 148017917 148018102 186 browser details YourSeq 88 328 891 2000 81.1% chr2 + 154626082 154626620 539 browser details YourSeq 86 353 527 2000 77.5% chr7 + 6162104 6162281 178 browser details YourSeq 84 424 533 2000 89.1% chr6 - 143039334 143177671 138338 browser details YourSeq 80 397 535 2000 79.2% chr11 - 100877584 100877732 149 browser details YourSeq 78 315 531 2000 91.5% chr6 - 148691136 148691374 239 browser details YourSeq 77 315 535 2000 83.0% chr7 - 49564791 49564995 205 browser details YourSeq 76 311 490 2000 80.5% chr13 - 43023796 43023985 190 browser details YourSeq 76 308 535 2000 79.3% chr1 + 71741775 71742106 332 browser details YourSeq 75 38 527 2000 70.3% chr5 - 96901661 96901841 181 browser details YourSeq 75 418 527 2000 88.7% chr2 - 72552521 72552710 190 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Med13 mediator complex subunit 13 [ Mus musculus (house mouse) ] Gene ID: 327987, updated on 10-Oct-2019 Gene summary Official Symbol Med13 provided by MGI Official Full Name mediator complex subunit 13 provided by MGI Primary source MGI:MGI:3029632 See related Ensembl:ENSMUSG00000034297 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 Thrap1; Trap240; D030023K18; 1110067M05Rik Expression Ubiquitous expression in thymus adult (RPKM 6.7), whole brain E14.5 (RPKM 6.6) and 28 other tissues See more Orthologs human all Genomic context Location: 11; 11 C See Med13 in Genome Data Viewer Exon count: 30 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (86267033..86357596, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (86079217..86171027, complement) Chromosome 11 - NC_000077.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Med13 ENSMUSG00000034297 Description mediator complex subunit 13 [Source:MGI Symbol;Acc:MGI:3029632] Gene Synonyms 1110067M05Rik, Thrap1, Trap240 Location Chromosome 11: 86,267,033-86,357,602 reverse strand. GRCm38:CM001004.2 About this gene This gene has 4 transcripts (splice variants), 264 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Med13-201 ENSMUST00000043624.8 10546 2171aa ENSMUSP00000044268.8 Protein coding CCDS36267 Q5SWW4 TSL:2 GENCODE basic APPRIS P1 Med13-203 ENSMUST00000140514.1 738 No protein - Retained intron - - TSL:3 Med13-204 ENSMUST00000144983.1 481 No protein - Retained intron - - TSL:2 Med13-202 ENSMUST00000136622.7 540 No protein - lncRNA - - TSL:5 110.57 kb Forward strand 86.26Mb 86.28Mb 86.30Mb 86.32Mb 86.34Mb 86.36Mb Genes Brip1os-202 >lncRNA Gm25427-201 >misc RNA (Comprehensive set... Contigs < AL592065.7 AL596256.14 > Genes (Comprehensive set... < Ints2-201protein coding< Med13-202lncRNA < Med13-204retained intron < Ints2-207retained intron < Med13-203retained intron < Ints2-209protein coding < Ints2-205protein coding < Ints2-208protein coding < Ints2-204retained intron < Ints2-210lncRNA < Med13-201protein coding Regulatory Build 86.26Mb 86.28Mb 86.30Mb 86.32Mb 86.34Mb 86.36Mb Reverse strand 110.57 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 RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000043624 < Med13-201protein coding Reverse strand 90.57 kb ENSMUSP00000044... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Mediator complex, subunit Med13, N-terminal, metazoa/fungi MID domain of medPIWI Mediator complex subunit Med13, C-terminal PANTHER PTHR10791:SF51 PTHR10791 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 1800 2171 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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