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Mouse Pdlim5 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Pdlim5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Pdlim5 gene (NCBI Reference Sequence: NM_001190852 ; Ensembl: ENSMUSG00000028273 ) is located on Mouse chromosome 3. 15 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 15 (Transcript: ENSMUST00000195975). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pdlim5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-147O20 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 a knock-out allele exhibit impaired cardiac muscle contractility, wider Z-lines, and dilated cardiomyopathy. Mice heterozygous for a gene trap allele exhibit impaired response to methamphetamine.

Exon 2 starts from about 5.27% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 3735 bp, and the size of intron 2 for 3'-loxP site insertion: 38827 bp. The size of effective cKO region: ~652 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 2 15 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pdlim5 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(7152bp) | A(28.5% 2038) | C(17.09% 1222) | T(34.23% 2448) | G(20.19% 1444)

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% chr3 - 142353136 142356135 3000 browser details YourSeq 40 53 98 3000 88.9% chr3 - 136492741 136492785 45 browser details YourSeq 36 394 439 3000 89.2% chr3 + 123002293 123002338 46 browser details YourSeq 35 24 235 3000 97.5% chr11 - 66183130 66183343 214 browser details YourSeq 33 2159 2213 3000 92.2% chr1 - 27813940 27813994 55 browser details YourSeq 32 388 429 3000 88.1% chr17 - 72718254 72718295 42 browser details YourSeq 27 394 428 3000 88.6% chr16 - 65444185 65444219 35

Note: The 3000 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 3000 1 3000 3000 100.0% chr3 - 142349484 142352483 3000 browser details YourSeq 241 26 326 3000 92.4% chr15 + 73161263 73476152 314890 browser details YourSeq 226 13 326 3000 93.9% chr1 + 119539863 119701539 161677 browser details YourSeq 216 26 324 3000 91.9% chr18 - 35493340 35493879 540 browser details YourSeq 203 14 326 3000 92.2% chr16 - 13769922 13770236 315 browser details YourSeq 199 38 323 3000 88.6% chr15 + 48967746 48968027 282 browser details YourSeq 198 13 323 3000 88.8% chr13 - 8997236 8997561 326 browser details YourSeq 188 78 326 3000 93.2% chr10 - 128148697 128159917 11221 browser details YourSeq 183 13 326 3000 93.0% chr17 + 35422448 35422948 501 browser details YourSeq 181 55 326 3000 94.2% chr12 - 84629077 84629558 482 browser details YourSeq 172 63 326 3000 93.1% chr2 - 135477760 135478132 373 browser details YourSeq 170 13 326 3000 93.0% chr13 - 63983543 63984099 557 browser details YourSeq 170 38 326 3000 92.5% chr11 + 78042224 78042549 326 browser details YourSeq 168 42 322 3000 93.4% chr17 + 29525004 29525287 284 browser details YourSeq 147 38 326 3000 86.1% chr11 - 85275808 85275982 175 browser details YourSeq 141 174 326 3000 97.4% chr2 - 80730518 80730670 153 browser details YourSeq 141 165 326 3000 95.0% chr17 - 12954851 12955027 177 browser details YourSeq 141 72 324 3000 93.3% chr13 + 54538367 54538840 474 browser details YourSeq 140 168 325 3000 95.5% chr10 - 73021783 73021949 167 browser details YourSeq 139 10 326 3000 84.6% chr3 - 96921752 96921906 155

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Pdlim5 PDZ and LIM domain 5 [ Mus musculus (house mouse) ] Gene ID: 56376, updated on 12-Aug-2019

Gene summary

Official Symbol Pdlim5 provided by MGI Official Full Name PDZ and LIM domain 5 provided by MGI Primary source MGI:MGI:1927489 See related Ensembl:ENSMUSG00000028273 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 Enh; LIM; Enh1; Enh2; Enh3; C87059; AI987914; 1110001A05Rik Expression Broad expression in heart adult (RPKM 41.4), bladder adult (RPKM 16.5) and 27 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 H1 See Pdlim5 in Genome Data Viewer

Exon count: 20

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (142239585..142395719, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (141903568..142058630, complement)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 13 transcripts

Gene: Pdlim5 ENSMUSG00000028273

Description PDZ and LIM domain 5 [Source:MGI Symbol;Acc:MGI:1927489] Gene Synonyms 1110001A05Rik, Enh, Enh2, Enh3 Location Chromosome 3: 142,239,590-142,395,696 reverse strand. GRCm38:CM000996.2 About this gene This gene has 13 transcripts (splice variants), 263 orthologues, 8 paralogues, is a member of 2 Ensembl protein families and is associated with 11 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pdlim5- ENSMUST00000029941.15 5016 591aa ENSMUSP00000029941.9 Protein CCDS17875 Q8CI51 TSL:1 201 coding GENCODE basic APPRIS P3

Pdlim5- ENSMUST00000170361.8 2010 239aa ENSMUSP00000128752.2 Protein CCDS38656 E9Q8P5 TSL:5 205 coding GENCODE basic

Pdlim5- ENSMUST00000195975.4 1845 614aa ENSMUSP00000142737.1 Protein CCDS80038 D9J302 TSL:1 206 coding GENCODE basic APPRIS ALT2

Pdlim5- ENSMUST00000168967.8 1755 234aa ENSMUSP00000132647.3 Protein CCDS80039 D9J303 TSL:1 204 coding GENCODE basic

Pdlim5- ENSMUST00000196220.4 1725 574aa ENSMUSP00000142460.1 Protein CCDS80037 D9J301 TSL:1 208 coding GENCODE basic APPRIS ALT2

Pdlim5- ENSMUST00000090134.11 1722 214aa ENSMUSP00000087595.5 Protein CCDS57258 Q9CRA2 TSL:1 203 coding GENCODE basic

Pdlim5- ENSMUST00000200043.4 1581 526aa ENSMUSP00000143343.1 Protein CCDS80036 D9J300 TSL:1 212 coding GENCODE basic APPRIS ALT2

Pdlim5- ENSMUST00000198381.4 1555 482aa ENSMUSP00000142899.1 Protein CCDS80035 D9J2Z9 TSL:5 211 coding GENCODE basic APPRIS ALT2

Pdlim5- ENSMUST00000196908.4 1196 337aa ENSMUSP00000143098.1 Protein CCDS17876 Q8CI51 TSL:1 209 coding GENCODE basic

Pdlim5- ENSMUST00000058626.8 873 239aa ENSMUSP00000059267.8 Protein - F8WJI6 TSL:5 202 coding GENCODE basic

Pdlim5- ENSMUST00000197808.4 759 253aa ENSMUSP00000142790.1 Protein - A0A0G2JEJ0 CDS 5' and 3' 210 coding incomplete TSL:3

Pdlim5- ENSMUST00000200650.4 669 223aa ENSMUSP00000143762.1 Protein - A0A0G2JGZ5 CDS 5' and 3' 213 coding incomplete TSL:5

Pdlim5- ENSMUST00000196050.1 2089 No - Retained - - TSL:NA 207 protein intron

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176.11 kb Forward strand 142.25Mb 142.30Mb 142.35Mb 142.40Mb Contigs < AC139128.6 AC158581.2 >

Genes (Comprehensive set... < Pdlim5-201protein coding

< Pdlim5-211protein coding

< Pdlim5-208protein coding

< Pdlim5-212protein coding

< Pdlim5-206protein coding

< Pdlim5-207retained intron < Pdlim5-205protein coding

< Pdlim5-213protein coding

< Pdlim5-210protein coding

< Pdlim5-203protein coding

< Pdlim5-204protein coding

< Pdlim5-209protein coding

< Pdlim5-202protein coding

Regulatory Build

142.25Mb 142.30Mb 142.35Mb 142.40Mb Reverse strand 176.11 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

processed transcript

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Transcript: ENSMUST00000195975

< Pdlim5-206protein coding

Reverse strand 149.13 kb

ENSMUSP00000142... MobiDB lite Low complexity (Seg) Superfamily PDZ superfamily SSF57716 SMART PDZ domain Zinc finger, LIM-type Pfam PDZ domain Domain of unknown function DUF4749 Zinc finger, LIM-type

PROSITE profiles PDZ domain Zinc finger, LIM-type PROSITE patterns Zinc finger, LIM-type PANTHER PTHR24214

PTHR24214:SF32 Gene3D 2.30.42.10 2.10.110.10 CDD cd00992 cd09453

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

Variant Legend frameshift variant missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 614

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

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