Mouse Mapk14 Conditional Knockout Project (CRISPR/Cas9)

http://www.alphaknockout.com/ Mouse Mapk14 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mapk14 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mapk14 gene ( NCBI Reference Sequence: NM_001168508 ; Ensembl: ENSMUSG00000053436 ) is located on mouse chromosome 17. 12 exons are identified , with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000004990). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the mouse Mapk14 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-369C13 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: Mice homozygous for various null mutations are embryonic to perinatal lethal showing multiple organ system defects. Mice homozygous for a knock-out mutation exhibit abnormal myoblast differentiation and delayed myofiber growth and maturation.

The knockout of Exon 3 will result in frameshift of the gene, and covers 5.46% of the coding region. The size of intron 2 for 5'-loxP site insertion: 1479 bp, and the size of intron 3 for 3'-loxP site insertion: 9302 bp. The size of effective cKO region: ~1500 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level.

Page 1 of 8 http://www.alphaknockout.com/

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 3 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Mapk14 Homology arm cKO region loxP site

Page 2 of 8 http://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. 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(7059bp) | A(26.22% 1851) | C(19.99% 1411) | G(25.06% 1769) | T(28.73% 2028)

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 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% chr17 + 28712134 28715133 3000 browser details YourSeq 149 1407 1584 3000 94.7% chr13 - 62812446 62812635 190 browser details YourSeq 149 1418 1978 3000 84.2% chr5 + 110035337 110035521 185 browser details YourSeq 149 1400 1575 3000 94.5% chr17 + 24145765 24145939 175 browser details YourSeq 146 1412 1590 3000 94.7% chr13 + 79554408 79554592 185 browser details YourSeq 145 1413 1584 3000 95.1% chr3 - 47865335 47865506 172 browser details YourSeq 145 1406 1585 3000 92.2% chr1 - 74553859 74554036 178 browser details YourSeq 144 1411 1584 3000 95.2% chr3 - 93685953 93686132 180 browser details YourSeq 144 1411 1587 3000 94.0% chr15 - 82009757 82009934 178 browser details YourSeq 143 1410 1583 3000 95.1% chr7 - 91454918 91455091 174 browser details YourSeq 143 1410 1580 3000 95.1% chr17 - 19328310 19328484 175 browser details YourSeq 143 1407 1583 3000 90.5% chr1 + 13056804 13056969 166 browser details YourSeq 142 1407 1586 3000 94.0% chr13 + 57677229 57677414 186 browser details YourSeq 142 1410 1583 3000 95.0% chr11 + 68896064 68896297 234 browser details YourSeq 140 1417 1584 3000 95.0% chr3 - 54623284 54623452 169 browser details YourSeq 140 1413 1584 3000 94.4% chr2 - 34905886 34906065 180 browser details YourSeq 140 1412 1582 3000 95.0% chr18 - 66137198 66137611 414 browser details YourSeq 140 1404 1584 3000 90.1% chr17 + 46149465 46149626 162 browser details YourSeq 140 1407 1580 3000 90.1% chr17 + 35128196 35128365 170 browser details YourSeq 140 1405 1584 3000 93.9% chr11 + 84170995 84171191 197

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% chr17 + 28715693 28718692 3000 browser details YourSeq 182 1196 1571 3000 86.6% chr1 + 93735934 93736310 377 browser details YourSeq 181 1196 1561 3000 89.6% chr2 - 121909624 121909998 375 browser details YourSeq 175 1196 1571 3000 92.5% chr11 - 119336991 119337475 485 browser details YourSeq 172 1146 1572 3000 86.5% chr11 + 80717484 80717982 499 browser details YourSeq 170 1240 1564 3000 86.9% chr11 - 120934204 120934529 326 browser details YourSeq 170 1141 1540 3000 89.1% chr8 + 23378725 23379131 407 browser details YourSeq 163 1141 1559 3000 90.6% chr5 + 120496420 120496873 454 browser details YourSeq 161 1141 1571 3000 90.6% chr5 + 124850009 124850456 448 browser details YourSeq 159 1138 1564 3000 90.2% chr17 - 12500646 12501109 464 browser details YourSeq 158 1138 1557 3000 90.3% chr3 + 68885304 68885726 423 browser details YourSeq 156 1144 1582 3000 89.6% chr15 - 93114403 93115014 612 browser details YourSeq 154 1141 1562 3000 82.2% chr2 + 12243635 12244039 405 browser details YourSeq 146 1240 1569 3000 87.2% chr7 + 127010545 127010871 327 browser details YourSeq 144 1141 1562 3000 89.9% chr10 - 79635557 79635976 420 browser details YourSeq 143 1196 1564 3000 89.2% chr18 + 48232630 48233056 427 browser details YourSeq 142 1253 1570 3000 86.3% chr10 - 43116765 43117275 511 browser details YourSeq 140 1219 1560 3000 84.4% chr5 - 33937651 33937988 338 browser details YourSeq 139 1240 1571 3000 89.1% chr1 - 172385099 172789427 404329 browser details YourSeq 138 1196 1564 3000 83.7% chr9 + 108453244 108453606 363

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 http://www.alphaknockout.com/ Gene and protein information: Mapk14 mitogen-activated protein kinase 14 [ Mus musculus (house mouse) ] Gene ID: 26416, updated on 1-Mar-2021

Gene summary

Official Symbol Mapk14 provided by MGI Official Full Name mitogen-activated protein kinase 14 provided by MGI Primary source MGI:MGI:1346865 See related Ensembl:ENSMUSG00000053436 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 p3; CSB; Crk; Mxi; p38; Crk1; Mxi2; p38-; p38M; p38a; CSBP2; Csbp1; PRKM14; PRKM15; p38MAPK; p38alpha; p38- Expression alpha Orthologs Ubiquitous expression in spleen adult (RPKM 35.2), thymus adult (RPKM 34.4) and 28 other tissues See more human all NEW Try the new Gene table Try the new Transcript table

Genomic context

Location: 17 A3.3; 17 14.85 cM See Mapk14 in Genome Data Viewer

Exon count: 15

Annotation release Status Assembly Chr Location

109 current GRCm39 (GCF_000001635.27) 17 NC_000083.7 (28910243..28967379)

108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (28691342..28748405)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (28828287..28885350)

Chromosome 17 - NC_000083.7

Page 5 of 8 http://www.alphaknockout.com/

Transcript information: This gene has 10 transcripts

Gene: Mapk14 ENSMUSG00000053436

Description mitogen-activated protein kinase 14 [Source:MGI Symbol;Acc:MGI:1346865] Gene Synonyms CSBP2, Crk1, Csbp1, Mxi2, p38, p38 MAP Kinase, p38 MAP kinase alpha, p38 MAPK, p38 alpha, p38-alpha, p38MAPK, p38a, p38alpha Location Chromosome 17: 28,910,303-28,967,380 forward strand. GRCm39:CM001010.3 About this gene This gene has 10 transcripts (splice variants), 294 orthologues, 19 paralogues and is associated with 49 phenotypes. Transcripts

UniProt Name Transcript ID bp Protein Translation ID Biotype CCDS Flags Match

Mapk14- ENSMUST00000114754.8 3592 283aa ENSMUSP00000110402.2 Protein coding CCDS50049 P47811-4 TSL:1 204 GENCODE basic

Mapk14- ENSMUST00000004990.14 3562 360aa ENSMUSP00000004990.7 Protein coding CCDS50048 P47811-3 TSL:1 201 GENCODE basic APPRIS ALT1

Mapk14- ENSMUST00000062694.16 3548 360aa ENSMUSP00000061958.9 Protein coding CCDS28583 P47811-1 TSL:1 202 GENCODE basic APPRIS P3

Mapk14- ENSMUST00000114752.3 3148 283aa ENSMUSP00000110400.2 Protein coding CCDS50049 P47811-4 TSL:1 203 GENCODE basic

Mapk14- ENSMUST00000114758.9 1478 258aa ENSMUSP00000110406.2 Protein coding - P47811-2 TSL:1 205 GENCODE basic

Mapk14- ENSMUST00000233250.2 1066 307aa ENSMUSP00000156692.2 Protein coding - A0A3B2WB60 GENCODE 209 basic

Mapk14- ENSMUST00000124886.9 866 227aa ENSMUSP00000116914.3 Protein coding - B2KF34 CDS 3' 206 incomplete TSL:3

Mapk14- ENSMUST00000233811.2 3490 50aa ENSMUSP00000156603.2 Nonsense mediated - A0A3B2WAZ7 - 210 decay

Mapk14- ENSMUST00000233095.2 223 No - Processed transcript - - - 208 protein

Mapk14- ENSMUST00000151613.2 509 No - Retained intron - - TSL:2 207 protein

Page 6 of 8 http://www.alphaknockout.com/

77.08 kb Forward strand

Genes (Comprehensive set... Mapk14-201 >protein coding

Mapk14-204 >protein coding

Mapk14-202 >protein coding

Mapk14-206 >protein coding Mapk14-207 >retained intron

Mapk14-210 >nonsense mediated decay

Mapk14-205 >protein coding

Mapk14-209 >protein coding

Mapk14-203 >protein coding

Gm4356-201 >protein coding

Mapk14-208 >processed transcript

Contigs < AC170998.2 Genes < Slc26a8-201protein coding (Comprehensive set...

< Slc26a8-205protein coding

< Slc26a8-202protein coding

< Slc26a8-206protein coding

< Slc26a8-208nonsense mediated decay

< Slc26a8-204processed transcript

Regulatory Build

Reverse strand 77.08 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

Page 7 of 8 http://www.alphaknockout.com/

Transcript: ENSMUST00000004990

57.08 kb Forward strand

Mapk14-201 >protein coding

ENSMUSP00000004... PDB-ENSP mappings Low complexity (Seg) Superfamily Protein kinase-like domain superfamily SMART Protein kinase domain Prints Mitogen-activated protein (MAP) kinase p38-like Pfam Protein kinase domain PROSITE profiles Protein kinase domain PROSITE patterns Mitogen-activated protein (MAP) kinase, conserved site

Protein kinase, ATP binding site PANTHER PTHR24055:SF110

PTHR24055 Gene3D 3.30.200.20

1.10.510.10 CDD Mitogen-activated protein kinase 14

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360

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

Page 8 of 8