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Mouse Plekho1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Plekho1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Plekho1 gene (NCBI Reference Sequence: NM_023320 ; Ensembl: ENSMUSG00000015745 ) is located on Mouse chromosome 3. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000015889). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Plekho1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-288N15 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 null allele exhibit age-dependent increase in bone volume and increased osteoblast activity.

Exon 6 covers 57.35% of the coding region. Start codon is in exon 1, and stop codon is in exon 6. The size of intron 5 for 5'-loxP site insertion: 1167 bp. The size of effective cKO region: ~1955 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 4 5 6 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Plekho1 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(7202bp) | A(24.72% 1780) | C(27.09% 1951) | T(22.44% 1616) | G(25.76% 1855)

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 - 95989884 95992883 3000 browser details YourSeq 122 2693 2856 3000 87.7% chr5 + 145111121 145111717 597 browser details YourSeq 122 2688 2870 3000 84.1% chr11 + 61628475 62040540 412066 browser details YourSeq 121 2694 2875 3000 84.9% chr3 - 69150343 69150533 191 browser details YourSeq 117 2694 2863 3000 85.9% chr15 + 79999346 79999516 171 browser details YourSeq 116 2694 2865 3000 83.8% chr1 + 192779613 192779784 172 browser details YourSeq 112 2694 2866 3000 82.6% chr11 + 76942180 76942357 178 browser details YourSeq 111 2694 2860 3000 83.0% chr11 - 101490657 101490821 165 browser details YourSeq 110 2697 2873 3000 81.3% chr13 - 105426886 105427068 183 browser details YourSeq 110 2726 2881 3000 85.7% chr12 - 102378913 102379066 154 browser details YourSeq 108 2726 2875 3000 86.6% chr6 + 94716728 94716891 164 browser details YourSeq 107 2726 2871 3000 87.5% chr10 + 13410551 13410715 165 browser details YourSeq 106 2729 2872 3000 87.4% chr9 - 31261079 31261222 144 browser details YourSeq 106 2695 2882 3000 90.9% chr19 - 15881674 15881870 197 browser details YourSeq 104 2694 2863 3000 84.8% chr9 - 55816863 55817031 169 browser details YourSeq 103 2723 2874 3000 88.8% chr4 + 133426656 133426820 165 browser details YourSeq 102 2717 2858 3000 86.7% chr17 - 53238451 53601209 362759 browser details YourSeq 102 2723 2875 3000 88.0% chr11 - 95921287 95921441 155 browser details YourSeq 101 2698 2861 3000 91.1% chr4 - 134685037 134685529 493 browser details YourSeq 101 2726 2869 3000 88.1% chr15 + 65780820 65780971 152

Note: The 3000 bp section upstream of Exon 6 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 - 95985932 95988931 3000 browser details YourSeq 34 1552 1590 3000 83.4% chr7 - 34835386 34835421 36 browser details YourSeq 30 21 148 3000 87.5% chr1 + 190162651 190162779 129 browser details YourSeq 28 1561 1588 3000 100.0% chr8 + 72307389 72307416 28 browser details YourSeq 27 1565 1591 3000 100.0% chr2 - 129103300 129103326 27 browser details YourSeq 27 1528 1556 3000 89.3% chr14 - 6472604 6472631 28 browser details YourSeq 27 1564 1590 3000 100.0% chr7 + 102261996 102262022 27 browser details YourSeq 26 1565 1590 3000 100.0% chr5 - 115461256 115461281 26 browser details YourSeq 26 1563 1590 3000 96.5% chr4 - 138316970 138316997 28 browser details YourSeq 26 1565 1590 3000 100.0% chr4 + 33134241 33134266 26 browser details YourSeq 25 97 121 3000 100.0% chr1 - 104480379 104480403 25 browser details YourSeq 24 88 112 3000 100.0% chr10 - 41914481 41914506 26 browser details YourSeq 23 1566 1590 3000 96.0% chr19 - 53322518 53322542 25 browser details YourSeq 23 1566 1590 3000 96.0% chr15 - 93394376 93394400 25 browser details YourSeq 23 1163 1186 3000 100.0% chr14 + 102585341 102585366 26 browser details YourSeq 21 322 342 3000 100.0% chr1 - 192528506 192528526 21

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Plekho1 pleckstrin homology domain containing, family O member 1 [ Mus musculus (house mouse) ] Gene ID: 67220, updated on 12-Aug-2019

Gene summary

Official Symbol Plekho1 provided by MGI Official Full Name pleckstrin homology domain containing, family O member 1 provided by MGI Primary source MGI:MGI:1914470 See related Ensembl:ENSMUSG00000015745 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Jza2; Ckip1; CKIP-1; JZA-20; 2810052M02Rik Expression Ubiquitous expression in bladder adult (RPKM 51.0), CNS E11.5 (RPKM 47.2) and 27 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 F2.1 See Plekho1 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (95988809..95999355, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (95792751..95799762, complement)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 5 transcripts

Gene: Plekho1 ENSMUSG00000015745

Description pleckstrin homology domain containing, family O member 1 [Source:MGI Symbol;Acc:MGI:1914470] Gene Synonyms 2810052M02Rik, CKIP-1, JZA-20, Jza2 Location Chromosome 3: 95,988,429-95,996,001 reverse strand. GRCm38:CM000996.2 About this gene This gene has 5 transcripts (splice variants), 242 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Plekho1- ENSMUST00000015889.9 1931 408aa ENSMUSP00000015889.3 Protein coding CCDS17627 Q9JIY0 TSL:1 201 GENCODE basic APPRIS P1

Plekho1- ENSMUST00000123006.7 1208 365aa ENSMUSP00000118665.1 Protein coding - F6XQM2 CDS 5' incomplete 202 TSL:5

Plekho1- ENSMUST00000130043.7 788 262aa ENSMUSP00000115035.1 Protein coding - F6VV25 CDS 5' and 3' 203 incomplete TSL:2

Plekho1- ENSMUST00000143485.1 441 124aa ENSMUSP00000114505.1 Protein coding - D3YVD1 CDS 3' incomplete 204 TSL:3

Plekho1- ENSMUST00000157043.1 362 No - Retained - - TSL:2 205 protein intron

27.57 kb Forward strand 95.98Mb 95.99Mb 96.00Mb Contigs AC092855.39 > Genes (Comprehensive set... < Plekho1-201protein coding < Vps45-201protein coding

< Plekho1-202protein coding

< Plekho1-203protein coding

< Plekho1-204protein coding

< Plekho1-205retained intron

Regulatory Build

95.98Mb 95.99Mb 96.00Mb Reverse strand 27.57 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000015889

< Plekho1-201protein coding

Reverse strand 7.57 kb

ENSMUSP00000015... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF50729 SMART Pleckstrin homology domain Pfam Pleckstrin homology domain PROSITE profiles Pleckstrin homology domain PANTHER Pleckstrin homology domain-containing family O member 1

PTHR15871 Gene3D PH-like domain superfamily CDD cd13317

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 408

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

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