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Mouse Med14 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Med14 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Med14 (NCBI Reference Sequence: NM_001048208 ; Ensembl: ENSMUSG00000064127 ) is located on Mouse X. 31 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 31 (Transcript: ENSMUST00000096495). Exon 16 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Med14 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-271H23 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: Male chimeras hemizygous for a gene trapped allele appear normal at E10.5.

Exon 16 starts from about 45.67% of the coding region. The knockout of Exon 16 will result in frameshift of the gene. The size of intron 15 for 5'-loxP site insertion: 2287 bp, and the size of intron 16 for 3'-loxP site insertion: 6890 bp. The size of effective cKO region: ~577 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 16 31 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Med14 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. 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(7077bp) | A(27.34% 1935) | C(19.19% 1358) | T(33.31% 2357) | G(20.16% 1427)

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% chrX - 12713910 12716909 3000 browser details YourSeq 239 1327 2046 3000 92.2% chr1 - 85464678 85588322 123645 browser details YourSeq 227 1321 2020 3000 87.6% chr6 + 87781443 87781798 356 browser details YourSeq 225 1320 2055 3000 91.3% chr10 - 93715539 93822651 107113 browser details YourSeq 219 1329 2020 3000 95.1% chr10 - 116856758 117373561 516804 browser details YourSeq 185 1404 2069 3000 84.1% chr18 + 42413134 42413470 337 browser details YourSeq 180 1407 2069 3000 85.0% chr5 - 147150116 147150547 432 browser details YourSeq 171 1405 2047 3000 84.4% chr1 + 59681457 59681725 269 browser details YourSeq 168 1437 2064 3000 89.8% chr7 - 19951123 19951725 603 browser details YourSeq 159 1412 2064 3000 82.2% chr8 + 70010000 70010225 226 browser details YourSeq 155 1879 2069 3000 93.9% chr3 + 94887908 94888437 530 browser details YourSeq 151 1892 2070 3000 94.2% chr7 - 116309108 116309292 185 browser details YourSeq 148 1889 2278 3000 82.9% chr2 - 165949038 165949214 177 browser details YourSeq 146 1443 2064 3000 88.7% chr2 - 168875977 168876582 606 browser details YourSeq 145 1892 2091 3000 92.4% chr1 - 130916897 130917166 270 browser details YourSeq 144 1889 2070 3000 87.5% chr1 - 181236564 181236731 168 browser details YourSeq 144 1892 2070 3000 92.1% chr4 + 129077462 129077638 177 browser details YourSeq 144 1891 2079 3000 94.6% chr12 + 70151846 70152059 214 browser details YourSeq 143 1892 2057 3000 95.0% chr7 - 82654524 82654694 171 browser details YourSeq 143 1892 2070 3000 94.5% chr5 + 147751074 147751253 180

Note: The 3000 bp section upstream of Exon 16 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% chrX - 12710333 12713332 3000 browser details YourSeq 66 1712 1851 3000 98.6% chr2 + 135300707 135301173 467 browser details YourSeq 55 1806 1861 3000 100.0% chr3 + 155514638 155515051 414 browser details YourSeq 47 1806 1852 3000 100.0% chr14 + 119812084 119812130 47 browser details YourSeq 46 1806 1851 3000 100.0% chrX - 114316309 114316354 46 browser details YourSeq 46 1806 1851 3000 100.0% chrX - 66170479 66170524 46 browser details YourSeq 46 1806 1851 3000 100.0% chr9 - 113609049 113609094 46 browser details YourSeq 46 1806 1851 3000 100.0% chr9 - 7028549 7028594 46 browser details YourSeq 46 1806 1851 3000 100.0% chr7 - 128172219 128172264 46 browser details YourSeq 46 1806 1851 3000 100.0% chr6 - 138287014 138287059 46 browser details YourSeq 46 1806 1851 3000 100.0% chr5 - 93360594 93360639 46 browser details YourSeq 46 1806 1851 3000 100.0% chr3 - 131227433 131227478 46 browser details YourSeq 46 1806 1851 3000 100.0% chr3 - 72984329 72984374 46 browser details YourSeq 46 1806 1851 3000 100.0% chr2 - 114866316 114866361 46 browser details YourSeq 46 1806 1851 3000 100.0% chr2 - 60543775 60543820 46 browser details YourSeq 46 1806 1851 3000 100.0% chr2 - 45567716 45567761 46 browser details YourSeq 46 1806 1851 3000 100.0% chr2 - 36380553 36380598 46 browser details YourSeq 46 1806 1851 3000 100.0% chr2 - 9612434 9612479 46 browser details YourSeq 46 1806 1851 3000 100.0% chr19 - 38896294 38896339 46 browser details YourSeq 46 1806 1851 3000 100.0% chr19 - 32401158 32401203 46

Note: The 3000 bp section downstream of Exon 16 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Med14 complex subunit 14 [ Mus musculus (house mouse) ] Gene ID: 26896, updated on 12-Aug-2019

Gene summary

Official Symbol Med14 provided by MGI Official Full Name mediator complex subunit 14 provided by MGI Primary source MGI:MGI:1349442 See related Ensembl:ENSMUSG00000064127 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 ORF1; Crsp2; Gm641; Trap170; AU041628; 9930001L01Rik Expression Ubiquitous expression in thymus adult (RPKM 13.6), whole brain E14.5 (RPKM 7.6) and 28 other tissues See more Orthologs human all

Genomic context

Location: X; X A1.1 See Med14 in Genome Data Viewer

Exon count: 33

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (12675368..12762594, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (12252497..12339099, complement)

Chromosome X - NC_000086.7

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Transcript information: This gene has 5 transcripts

Gene: Med14 ENSMUSG00000064127

Description mediator complex subunit 14 [Source:MGI Symbol;Acc:MGI:1349442] Gene Synonyms 9930001L01Rik, Crsp2, ENSMUSG00000073278, LOC270579, ORF1, Trap170 Location Chromosome X: 12,675,369-12,762,073 reverse strand. GRCm38:CM001013.2 About this gene This gene has 5 transcripts (splice variants), 207 orthologues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Med14-202 ENSMUST00000096495.10 6963 1459aa ENSMUSP00000094239.4 Protein coding CCDS40874 A2ABV5 TSL:5 GENCODE basic APPRIS P2

Med14-203 ENSMUST00000115481.7 3729 798aa ENSMUSP00000111143.1 Protein coding - A2BDP0 TSL:1 GENCODE basic

Med14-201 ENSMUST00000076016.5 2561 700aa ENSMUSP00000075395.5 Protein coding - A2BDN7 TSL:1 GENCODE basic APPRIS ALT2

Med14-204 ENSMUST00000124053.1 5129 No protein - Retained intron - - TSL:1

Med14-205 ENSMUST00000124070.1 627 No protein - Retained intron - - TSL:3

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106.70 kb Forward strand

12.68Mb 12.70Mb 12.72Mb 12.74Mb 12.76Mb Gm16265-201 >processed pseudogene Gm14634-201 >lncRNA (Comprehensive set...

Gm14634-202 >lncRNA

Contigs BX000537.11 > AL662925.12 > Genes (Comprehensive set... < 1810030O07Rik-201protein coding < Med14-205retained intron

< Med14-202protein coding

< Med14-203protein coding

< Med14-204retained intron

< Med14-201protein coding

Regulatory Build

12.68Mb 12.70Mb 12.72Mb 12.74Mb 12.76Mb Reverse strand 106.70 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene pseudogene processed transcript

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Transcript: ENSMUST00000096495

< Med14-202protein coding

Reverse strand 86.70 kb

ENSMUSP00000094... MobiDB lite Low complexity (Seg) Pfam Mediator complex, subunit Med14 PANTHER Mediator complex, subunit Med14

PTHR12809:SF2

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 1459

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

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