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Mouse Med24 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Med24 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Med24 (NCBI Reference Sequence: NM_011869 ; Ensembl: ENSMUSG00000017210 ) is located on Mouse 11. 26 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 26 (Transcript: ENSMUST00000017354). Exon 7~12 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Med24 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-262M10 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: Homozygous mutant mice die prior to birth exhibiting abnormal heart development, neural tube defects, and anemia.

Exon 7 starts from about 18.91% of the coding region. The knockout of Exon 7~12 will result in frameshift of the gene. The size of intron 6 for 5'-loxP site insertion: 1384 bp, and the size of intron 12 for 3'-loxP site insertion: 773 bp. The size of effective cKO region: ~2993 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 5 6 7 8 9 10 11 12 13 14 26 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Med24 Homology arm cKO region loxP site

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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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(9493bp) | A(21.15% 2008) | C(25.78% 2447) | T(26.65% 2530) | G(26.42% 2508)

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.

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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 - 98716776 98719775 3000 browser details YourSeq 119 111 366 3000 91.8% chr10 - 88247878 88248131 254 browser details YourSeq 119 212 366 3000 92.2% chr12 + 34947113 34947269 157 browser details YourSeq 115 228 369 3000 93.9% chr1 - 167359648 167359795 148 browser details YourSeq 114 217 361 3000 91.4% chr1 + 74183402 74258584 75183 browser details YourSeq 113 224 365 3000 90.3% chr5 - 100465649 100465789 141 browser details YourSeq 113 207 366 3000 85.5% chr11 + 69507634 69507783 150 browser details YourSeq 112 227 378 3000 92.4% chr7 - 35441333 35441496 164 browser details YourSeq 112 212 361 3000 92.5% chr11 + 95850735 95850890 156 browser details YourSeq 111 228 371 3000 90.6% chr2 - 69246641 69246788 148 browser details YourSeq 111 228 372 3000 92.4% chr3 + 95966729 95966876 148 browser details YourSeq 111 224 369 3000 89.4% chr16 + 28745619 28745762 144 browser details YourSeq 110 240 369 3000 94.4% chr2 - 71870800 71870933 134 browser details YourSeq 110 237 366 3000 93.1% chr11 - 8114738 8262500 147763 browser details YourSeq 109 228 369 3000 93.0% chr6 - 38000364 38000508 145 browser details YourSeq 109 224 369 3000 88.0% chr10 - 93647547 93647690 144 browser details YourSeq 109 209 369 3000 89.8% chr1 - 153192769 153192938 170 browser details YourSeq 109 223 369 3000 90.4% chr15 + 93338702 93338851 150 browser details YourSeq 108 228 367 3000 92.2% chr1 - 39906516 39906658 143 browser details YourSeq 108 228 375 3000 89.7% chr8 + 107427444 107427595 152

Note: The 3000 bp section upstream of Exon 7 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 - 98710783 98713782 3000 browser details YourSeq 246 1655 2116 3000 89.2% chr7 - 80262569 80263017 449 browser details YourSeq 240 1658 2119 3000 93.8% chr6 - 149076206 149076688 483 browser details YourSeq 231 1508 1882 3000 95.7% chr2 - 156300217 156300863 647 browser details YourSeq 230 1642 2063 3000 94.2% chr2 + 30201484 30202096 613 browser details YourSeq 229 1470 1847 3000 93.3% chr3 - 135497774 135498304 531 browser details YourSeq 229 1470 2085 3000 89.4% chr2 + 167624150 167624621 472 browser details YourSeq 228 1653 2070 3000 95.6% chr4 + 103255809 103256379 571 browser details YourSeq 215 1642 1885 3000 97.9% chrX - 129554531 129712925 158395 browser details YourSeq 210 1472 1882 3000 96.9% chr6 + 88947416 88947841 426 browser details YourSeq 210 1474 1847 3000 91.9% chr3 + 37468714 37469027 314 browser details YourSeq 209 1470 1847 3000 96.1% chr5 - 103751919 103752296 378 browser details YourSeq 206 1638 1886 3000 92.8% chr13 + 67351696 67351922 227 browser details YourSeq 205 1470 1882 3000 93.9% chrX - 144326671 144327052 382 browser details YourSeq 205 1656 1887 3000 96.9% chr10 - 128898279 128898549 271 browser details YourSeq 204 1657 1886 3000 95.4% chr10 - 75956704 75956929 226 browser details YourSeq 204 1649 1886 3000 93.0% chr8 + 88187428 88187644 217 browser details YourSeq 203 1656 1882 3000 97.3% chr5 + 143024540 143024771 232 browser details YourSeq 202 1658 1882 3000 94.4% chr9 - 75450896 75451110 215 browser details YourSeq 202 1643 1882 3000 93.6% chr6 + 19626702 19626933 232

Note: The 3000 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Med24 complex subunit 24 [ Mus musculus (house mouse) ] Gene ID: 23989, updated on 24-Oct-2019

Gene summary

Official Symbol Med24 provided by MGI Official Full Name mediator complex subunit 24 provided by MGI Primary source MGI:MGI:1344385 See related Ensembl:ENSMUSG00000017210 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Gse2; 911GSE; Pparb2; R75526; Thrap4; DRIP100; Pparbp2; Trap100; AU040102; AW547152; D11Ertd307e Summary This gene encodes a component of the mediator complex (also known as TRAP, SMCC, DRIP, or ARC), a transcriptional Expression coactivator complex thought to be required for the expression of almost all . The mediator complex is recruited by transcriptional activators or nuclear receptors to induce gene expression, possibly by interacting with RNA polymerase II and promoting the formation of a transcriptional pre-initiation complex. The product of this gene may form a submodule of the mediator complex that magnifies the effects of activators on the general transcription machinery. Alternatively spliced transcript variants of this gene have been described, but their full-length nature is not known. [provided by RefSeq, Jul 2008] Orthologs Ubiquitous expression in thymus adult (RPKM 41.8), testis adult (RPKM 39.3) and 28 other tissues See more human all

Genomic context

Location: 11 D; 11 62.46 cM See Med24 in Genome Data Viewer Exon count: 28

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (98704591..98729453, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (98565905..98590749, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 12 transcripts

Gene: Med24 ENSMUSG00000017210

Description mediator complex subunit 24 [Source:MGI Symbol;Acc:MGI:1344385] Gene Synonyms 100kDa, D11Ertd307e, DRIP100, Gse2, Pparb2, R75526, Thrap4, Trap100 Location Chromosome 11: 98,704,591-98,729,435 reverse strand. GRCm38:CM001004.2 About this gene This gene has 12 transcripts (splice variants), 212 orthologues, is a member of 1 Ensembl protein family and is associated with 16 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Med24- ENSMUST00000017354.12 3410 987aa ENSMUSP00000017354.6 Protein coding CCDS25361 Q99K74 TSL:1 201 GENCODE basic APPRIS P2

Med24- ENSMUST00000100500.8 3133 1006aa ENSMUSP00000098069.2 Protein coding - A6PW47 TSL:5 202 GENCODE basic APPRIS ALT2

Med24- ENSMUST00000126565.1 416 90aa ENSMUSP00000118820.1 Protein coding - A6PW46 CDS 3' 205 incomplete TSL:5

Med24- ENSMUST00000138750.7 3165 70aa ENSMUSP00000120002.1 Nonsense mediated - F6XX22 TSL:1 207 decay

Med24- ENSMUST00000125064.7 4577 No - Retained intron - - TSL:1 204 protein

Med24- ENSMUST00000137328.7 2830 No - Retained intron - - TSL:1 206 protein

Med24- ENSMUST00000156378.1 760 No - Retained intron - - TSL:5 212 protein

Med24- ENSMUST00000144720.1 685 No - Retained intron - - TSL:2 211 protein

Med24- ENSMUST00000144048.1 537 No - Retained intron - - TSL:1 210 protein

Med24- ENSMUST00000124371.1 478 No - Retained intron - - TSL:3 203 protein

Med24- ENSMUST00000141566.1 453 No - lncRNA - - TSL:3 209 protein

Med24- ENSMUST00000139849.1 370 No - lncRNA - - TSL:3 208 protein

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44.84 kb Forward strand 98.70Mb 98.71Mb 98.72Mb 98.73Mb Genes Psmd3-201 >protein codingCsf3-201 >protein coding (Comprehensive set...

Psmd3-205 >retained intron

Contigs < AL590963.11 Genes (Comprehensive set... < Med24-201protein coding

< Med24-204retained intron

< Med24-212retained intron < Med24-209lncRNA

< Med24-202protein coding

< Med24-207nonsense mediated decay

< Med24-203retained intron < Med24-205protein coding

< Med24-208lncRNA < Med24-210retained intron

< Med24-206retained intron

< Med24-211retained intron

< Gm22059-201snoRNA

Regulatory Build

98.70Mb 98.71Mb 98.72Mb 98.73Mb Reverse strand 44.84 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000017354

< Med24-201protein coding

Reverse strand 24.84 kb

ENSMUSP00000017... Low complexity (Seg) Pfam Mediator complex, subunit Med24,

PANTHER Mediator complex, subunit Med24,

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 987

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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