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Mouse Plekha8 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Plekha8 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Plekha8 gene (NCBI Reference Sequence: NM_001164361 ; Ensembl: ENSMUSG00000005225 ) is located on Mouse chromosome 6. 14 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000119706). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Plekha8 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-3I1 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:

Exon 5 starts from about 28.2% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2436 bp, and the size of intron 6 for 3'-loxP site insertion: 1808 bp. The size of effective cKO region: ~1619 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 5 6 7 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Plekha8 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(8119bp) | A(27.71% 2250) | C(21.51% 1746) | T(29.98% 2434) | G(20.8% 1689)

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% chr6 + 54615945 54618944 3000 browser details YourSeq 183 1454 1845 3000 93.4% chr1 + 155316055 155316627 573 browser details YourSeq 178 1463 1852 3000 91.2% chr18 + 12156935 12157380 446 browser details YourSeq 170 2305 2588 3000 78.2% chr9 + 44038065 44038340 276 browser details YourSeq 170 1447 1846 3000 92.1% chr5 + 66221740 66222359 620 browser details YourSeq 165 1653 1846 3000 92.8% chr4 - 14790579 14790782 204 browser details YourSeq 164 1662 1846 3000 94.6% chr8 - 57389709 57389897 189 browser details YourSeq 163 1665 1846 3000 95.1% chr6 - 140903251 140903437 187 browser details YourSeq 160 1660 1845 3000 93.1% chr3 + 133595841 133596026 186 browser details YourSeq 159 1665 1847 3000 93.5% chr5 - 100573807 100573989 183 browser details YourSeq 157 1662 1842 3000 94.0% chr3 - 8977053 8977235 183 browser details YourSeq 157 1662 1846 3000 92.5% chr2 - 33378348 33378532 185 browser details YourSeq 157 1661 1846 3000 92.5% chr1 + 151006272 151006458 187 browser details YourSeq 156 1665 1846 3000 94.4% chr16 - 93783208 93783394 187 browser details YourSeq 156 1665 1848 3000 94.4% chr14 + 73799215 73799403 189 browser details YourSeq 156 2195 2588 3000 78.7% chr14 + 58551616 58551986 371 browser details YourSeq 155 1665 1845 3000 91.7% chr18 - 78112627 78112806 180 browser details YourSeq 155 1665 1843 3000 93.3% chr6 + 84919906 84920084 179 browser details YourSeq 154 1665 1846 3000 90.6% chr19 - 5783560 5783739 180 browser details YourSeq 154 2195 2565 3000 81.0% chr18 - 46652079 46652429 351

Note: The 3000 bp section upstream of Exon 5 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% chr6 + 54620564 54623563 3000 browser details YourSeq 142 1561 1712 3000 96.8% chr17 + 32593533 32593684 152 browser details YourSeq 110 1328 1505 3000 86.0% chr5 - 132921672 132921849 178 browser details YourSeq 110 1328 1503 3000 90.5% chr11 - 82447301 82447494 194 browser details YourSeq 105 1328 1505 3000 88.0% chr1 + 66376899 66377075 177 browser details YourSeq 100 1333 1501 3000 83.3% chr1 - 159171596 159171806 211 browser details YourSeq 100 1349 1505 3000 85.9% chr3 + 157793910 157794082 173 browser details YourSeq 100 1328 1505 3000 88.5% chr12 + 55556035 55556232 198 browser details YourSeq 98 1363 1505 3000 85.5% chr4 - 131695664 131695807 144 browser details YourSeq 98 1328 1506 3000 79.6% chr5 + 104973438 104973616 179 browser details YourSeq 97 1363 1505 3000 84.8% chr6 - 58966647 58966789 143 browser details YourSeq 96 1328 1474 3000 88.1% chr7 + 126105450 126105598 149 browser details YourSeq 94 1328 1474 3000 90.5% chr5 - 32108841 32108988 148 browser details YourSeq 90 1328 1474 3000 89.5% chr9 - 32624096 32624259 164 browser details YourSeq 90 1327 1474 3000 85.1% chr3 - 121422180 121422327 148 browser details YourSeq 90 1328 1505 3000 89.4% chr14 + 76691562 76691777 216 browser details YourSeq 90 1358 1504 3000 86.5% chr1 + 150954334 150954477 144 browser details YourSeq 88 1331 1505 3000 82.8% chrX_GL456233_random + 24581 24755 175 browser details YourSeq 86 1328 1505 3000 83.1% chr3 - 127385787 127385980 194 browser details YourSeq 85 1385 1505 3000 86.3% chr8 + 45675587 45675707 121

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: Plekha8 pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 8 [ Mus musculus (house mouse) ] Gene ID: 231999, updated on 12-Aug-2019

Gene summary

Official Symbol Plekha8 provided by MGI Official Full Name pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 8 provided by MGI Primary source MGI:MGI:2681164 See related Ensembl:ENSMUSG00000005225 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 Fapp2; Fapp-2; AA517676; BC052360; 6330400G01 Expression Ubiquitous expression in CNS E11.5 (RPKM 6.6), colon adult (RPKM 5.8) and 27 other tissues See more Orthologs human all

Genomic context

Location: 6; 6 B3 See Plekha8 in Genome Data Viewer

Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (54595111..54645823)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (54545105..54595817)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 2 transcripts

Gene: Plekha8 ENSMUSG00000005225

Description pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 8 [Source:MGI Symbol;Acc:MGI:2681164] Gene Synonyms FAPP2 Location Chromosome 6: 54,595,111-54,645,839 forward strand. GRCm38:CM000999.2 About this gene This gene has 2 transcripts (splice variants), 213 orthologues, 5 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Plekha8-202 ENSMUST00000119706.7 6739 519aa ENSMUSP00000112466.1 Protein coding CCDS51781 Q80W71 TSL:2 GENCODE basic APPRIS P1

Plekha8-201 ENSMUST00000101385.2 1838 474aa ENSMUSP00000098935.2 Protein coding CCDS39491 Q80W71 TSL:1 GENCODE basic

70.73 kb Forward strand

54.60Mb 54.62Mb 54.64Mb Genes (Comprehensive set... Plekha8-202 >protein coding

Plekha8-201 >protein coding

Contigs AC084800.5 > Genes < Fkbp14-201protein codin

< Fkbp14-207nonsense mediated decay

< Fkbp14-202protein coding

< Fkbp14-203retained intron

< Fkbp14-204lncRNA

< Fkbp14-205retained intron

Regulatory Build

54.60Mb 54.62Mb 54.64Mb Reverse strand 70.73 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: ENSMUST00000119706

50.73 kb Forward strand

Plekha8-202 >protein coding

ENSMUSP00000112... MobiDB lite Low complexity (Seg) Superfamily SSF50729 Glycolipid transfer protein superfamily

SMART Pleckstrin homology domain

Pfam Pleckstrin homology domain Glycolipid transfer protein domain

PROSITE profiles Pleckstrin homology domain

PANTHER PTHR10219

PTHR10219:SF73 Gene3D PH-like domain superfamily Glycolipid transfer protein superfamily

CDD cd01247

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 519

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

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